Identification of critical stage responding to consecutive monoculture obstacle in Rehmannia glutinosa L.

  • Xiaoran Wang
  • Zhen Li
  • Lu Yang
  • Weixi Li
  • Yuhong Wang
  • Mingjie Li
  • Zhongyi ZhangEmail author
  • Xinjian ChenEmail author
Original Article


Rehmannia glutinosa L. (R. glutinosa) is one of the most common traditional Chinese medicines, whose productivity and quality, however, are seriously impacted by replanting disease, also known as consecutive monoculture obstacle. Little is known about R. glutinosa’s critical responsive stage to consecutive monoculture. There were two parts to this study: first, we analyzed different gene expression profiles in root of R. glutinosa between first-year planting (FP) and second-year replanting (SP) in five development stages (stage I to V) using Illumina sequencing to interpret the stage that most dramatically responding to consecutive monoculture; second, applying tissue culture technique, the harm of consecutive monoculture in a sterile condition was simulated and the medium was added with the soil extracts from FP and SP soil. The results showed that a significant difference in gene expression appeared at stage I under consecutive monoculture. Using a calcium indicator, the fluo-3 fluorescence to detect calcium distribution in the root tip of seedlings cultured in medium, the results revealed that after being cultured for 10 days, the SP fluorescence intensity of the seedlings was significantly higher than that of FP while crown position was particularly prominent. The evidence from gene expression profiling and tissue culture reached agreement that early stage of R. glutinosa growth was the critical stage to perceive consecutive monoculture stress. The results are of great significance to study the molecular mechanism of consecutive monoculture, which is expected to await a quick and accurate evaluation of land feasibility to plant R. glutinosa.


R. glutinosa Consecutive monoculture obstacle Digital gene expression Calcium signaling Aseptic seedling 


R. glutinosa

Rehmannia glutinosa L.


First-year planting


Second-year planting


High performance liquid chromatography

Stage I

From cropping R. glutinosa planted seedlings to the root length of 8 cm, June 10th

Stage II

The root length of 8–15 cm, July 1st

Stage III

The diameter column of 1–2 cm, July 29th

Stage IV

The diameter column of 2–4 cm August 26th

Stage V

Maturity at the end of September 30th


Digital gene expression profiling


Kyoto Encyclopedia of Genes and Genomes


Differentially expressed genes


Gene ontology


MS medium with extracts of rhizosphere soil in which R. glutinosa had never been replanted


MS medium with extracts of rhizosphere soil in which R. glutinosa had been replanted for 1 year


MS medium with extracts of rhizosphere soil in which R. glutinosa had been replanted for 2 years



This work was supported by grants from the National Natural Science Foundation of China (31271674), and Key Research Project of Colleges and Universities in Henan Province (17A180024).

Supplementary material

11738_2019_2847_MOESM1_ESM.tif (356 kb)
Fig. S1 The tuberous roots of R. glutinosa at five different developmental stages (I, II, III, IV and V) as sampling time (TIFF 356 kb)
11738_2019_2847_MOESM2_ESM.xlsx (11 kb)
Table S1 Primer sequences used to assay ten differentially expressed genes by qRT-PCR verification in FP and SP (XLSX 10 kb)
11738_2019_2847_MOESM3_ESM.xlsx (10 kb)
Table S2 Primer sequences for ten differentially expressed genes screened by DGE, which were enriched in at least one of 124 KEGG pathways, used to compare transcript abundance in aseptic seedlings cultured for 10 days in MENP (never replanted) and MEPO (replanted for one year) by qRT-PCR (XLSX 10 kb)
11738_2019_2847_MOESM4_ESM.tif (796 kb)
Fig. S2 The induction process of R. glutinosa aseptic seedlings. The aseptic pieces of root with bud eyes were cultured in 1/2 MS around 14 days. When regenerants reached 3-4 cm in length, they could be transferred to basic MS medium to continue nurturing (TIFF 795 kb)
11738_2019_2847_MOESM5_ESM.xlsx (10 kb)
Table S3 Summary of DGE tags of R. glutinosa in FP and SP at all five stages (XLSX 10 kb)
11738_2019_2847_MOESM6_ESM.tif (1.7 mb)
Fig. S3 Validation of the DGE results. Ten differentially expressed genes (five up-regulated and five down-regulated) between FP and SP were randomly selected to detect transcript abundance by qRT-PCR. Eight of the ten genes provided consistent trends, indicated that DGE results were credible (TIFF 1782 kb)
11738_2019_2847_MOESM7_ESM.xlsx (1.7 mb)
Table S4 The differentially expressed genes (DEGs) between FP and SP at stage I, of which transcript abundance was quantified in RPKM method, and annotation against KEGG, GO and Nr databases (XLSX 1691 kb)
11738_2019_2847_MOESM8_ESM.xlsx (98 kb)
Table S5 The details of GO ‘biological_process’ of differentially expressed genes in SP compared to FP at stage I (XLSX 98 kb)
11738_2019_2847_MOESM9_ESM.xlsx (24 kb)
Table S6 The details of GO ‘celluar_component’ enriched by differentially expressed genes in SP compared to FP at stage I (XLSX 24 kb)
11738_2019_2847_MOESM10_ESM.xlsx (47 kb)
Table S7 The details of GO ‘molecular_function’ enriched by differentially expressed genes in SP compared to FP at stage I (XLSX 46 kb)
11738_2019_2847_MOESM11_ESM.xlsx (18 kb)
Table S8 The details of KEGG pathways enriched by differentially expressed genes in SP compared to FP at stage I (XLSX 18 kb)
11738_2019_2847_MOESM12_ESM.tif (2.3 mb)
Fig. S4 The aseptic seedlings were cultured in MENP (never replanted), MEPO (replanted for one year), and MEPT (replanted for two years) for 30 days (TIFF 2333 kb)
11738_2019_2847_MOESM13_ESM.xlsx (13 kb)
Table S9 Fresh weight and root length of seedlings cultured in MENP (never replanted) and MEPO (replanted for one year) after 10, 15 and 25 days. Three biological replications were performed in measurement of each biomass (XLSX 12 kb)
11738_2019_2847_MOESM14_ESM.tif (334 kb)
Fig. S5 The phenotype of mature tuberous roots of R. glutinosa planted in FP and SP. Instead of elongation process, swelling of R. glutinosa roots has a dramatic inhibition under consecutive monoculture (TIFF 333 kb)


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Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2019

Authors and Affiliations

  1. 1.College of Life SciencesHenan Agricultural UniversityZhengzhouChina
  2. 2.College of Crop SciencesFujian Agriculture and Forestry UniversityFuzhouChina

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