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Identification of Bupleurum (Apiaceae) seeds by allele-specific PCR based on ITS sequences

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Abstract

The traditional Chinese medicine Bupleuri radix (chaihu) is the dried roots of Bupleurum chinense and Bupleurum scorzonerifolium and many adulterants exist because of the differences in traditional understanding, medication habits and seed resources. Therefore, rapid and accurate identification methods for Bupleurum (Apiaceae) seeds are required. We analyzed the internal transcribed spacer (ITS) sequences of five common Bupleurum species to detect variations in them, including B. chinense, B. scorzonerifolium, B. marginatum var. stenophyllum, B. falcatum and B. smithii var.parvifolium. Based on single nucleotide polymorphisms (SNPs) in the ITS region, we designed five specific primer pairs and used these primers in an allele-specific PCR technique to establish a robust molecular identification method. The neighbor-joining (NJ) tree of ITS sequences showed that five Bupleurum species formed their own monophyly. Five specific primer pairs were designed and integrated into a specific PCR master mix and cycling conditions. The primer pair of BCF/R8 for B. chinense could amplify a specific identification band of 429 bp and the minimum detection limit of the 5 g mixture was about 5%; BSF/R11 for B. scorzonerifolium could amplify a specific 464 bp band and the minimum detection limit was about 5%; BMSF/R1 for B. marginatum var.stenophyllum could amplify a specific 344 bp band and the minimum detection limit was about 1%; BFF/R7 for B. falcatum could amplify a specific 137 bp band and the minimum detection limit was about 1%; BSPF/R1 for B. smithii var. parvifolium could amplify a specific 390 bp band. Subsequent analysis proved the reliable accuracy and good practicability of the five specific identification primers, indicating that the allele-specific primer PCR identification method can quickly identify Bupleurum seeds. The method elaborated in this study has the advantages of simple operation, good accuracy and high efficiency.

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References

  • Guo WY, Niu YZ, Yao CY (2006) Advances in application of DNA molecular marking technology in quality identification of taditional chinese medicine. XinXiang YiXue Yuan XueBao 23(6):635–637

    CAS  Google Scholar 

  • Han BX, Yuan Y, Huang LQ, Zhao Q, Wang J (2017) Specific PCR identification between Peucedanum praeruptorum and Angelica decursiva and identification between them and adulterant using DNA barcode. Pharmacogn Mag 13(49):38–45

    PubMed  PubMed Central  Google Scholar 

  • Huang HP, Zhang YL, Gao GY (2017a) Problems and countermeasures of chinese herbal medicine seed and seedling industrial development in Bozhou. AnHui NongYe Ke Xue 45(11):240–241

    Google Scholar 

  • Huang HZ, Wang W, Fu H, Wang W, Yang SH (2017b) Research progress on medicinal plant resources of Bupleurum L. Zhong Cao Yao 48(14):2989–2996

    Google Scholar 

  • Kuang H, Sun S, Yang B, Xia Y, Feng W (2009) New megastigmane sesquiterpene and indole alkaloid glucosides from the aerial parts of Bupleurum chinense. DC. Fitoterapia 80(1):35–38

    CAS  PubMed  Google Scholar 

  • Li GL, Song YD, Lü ZH, Liu CH, Li J, Zhang XN (2016) Identification of Zizyphus jujuba with the counterfeit products by allele-specific PCR based on ITS genes sequence. ZhongGuo Xian Dai Zhong Yao 18(12):1566–1570

    Google Scholar 

  • Liang ZB (2012) Investigation of Medicinal Plant resources of Bupleurum and quality evaluation based on metabolomics. Dissertation, Southern Medical University

  • Liang HX, Wang W, Duan QY, Zhang W, Shang K, Zhang Y (2018) A new methodsbased on PCR for identification of Cordyceps sinensis. Zhong Yao Cai 41(03):546–550

    Google Scholar 

  • Liu J, Huang SM, Sun MY, Liu SY, Liu YM, Wang WX, Zhang XR, Wang HZ, Hua W (2012) An improved allele-specific PCR primer design method for SNP marker analysis and ITS application. Plant Methods 8(1):34

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liu XY, Wang L, Wang PF, Feng Y, Qiao YG, Wang JS (2016) Study on morphological and anatomical characteristics of Bupleurum chinense L. ShiZhen Guo YiGuo Yao 10:2420–2422

    Google Scholar 

  • Ma XG, Wang CB, He XJ (2010) Micromorphological features of pericarp surface of Bupleurum L. (Apiaceae) in china and Its taxonomic significance. Xibei Zhiwu Xuebao 30(7):1388–1396

    Google Scholar 

  • Mammadov J, Aggarwal R, Buyyarapu R, Kumpatla S (2012) SNP markers and their impact on plant breeding. Int. J. Plant Genom. 2012:1–11

    Google Scholar 

  • National Pharmacopoeia Commission (2015) Pharmacopoeia of the People's Republic of China (Part 1). China Medical Science and Technology Press, Beijing

    Google Scholar 

  • Peng YX, Zhang DJ, Wu WG, Mi YW, Lin HM (2018) Comparative analysis on seedling growth dynamics and physiological indices of Bupleurum smithii var. parvifolium and B. scorzonerifolium. China J Chin Mater Med 43(12):2489–2494

    Google Scholar 

  • Qin XM, Wang YQ, Yue JY (2005) Analysis of the status of cultivated Bupleurum resources. ZhongYao Yan Jiu Yu Xin Xi 7(8):30–32

    Google Scholar 

  • Wang CB, Ma XG, He XJ (2011) Fruit features of some Bupleurum species (Apiaceae) and their systematical implication. Plant Sci J 1(4):399–408

    Google Scholar 

  • Wang G, Cao P, Wei XM, Han JP (2019) Applications of molecular markers in study of germplasm resources of medicinal plants. ZhongGuo Xian Dai Zhong Yao 21(11):1435–1444

    Google Scholar 

  • Wei JH, Chen SL, Cheng HZ, Li MJ, Yang CM (2005) Seed & Seedling standardization project of chinese medicinal materials. World Sci Technol Mod Tradit Chin Med 7(6):104–108

    Google Scholar 

  • Xue Y, Li XW, Xing W, Zhang FS, Qin XM (2015) Identification of specific primers for Bupleurum chinense and Bupleurum Scorzonerifolium. ShiZhen Guo YiGuo Yao 26(02):370–372

    CAS  Google Scholar 

  • Yang YJ, Zheng W, Guo JQ, Fang W, Zhang DJ (2019) Comparative analysis on chemical constituents in Bupleurum chinense, B. marginatum, B. marginatum var stenophyllum and B. smithii var parvifolium. China J Chin Mater Med 44(2):332–337

    Google Scholar 

  • Yuan BC, Li WD, Ma YS, Zhou S, Liu Y (2017) The molecular identification of Bupleurum medicinal species and the quality investigation of Bupleuri radix. Acta Pharm Sin 52(1):162–171

    Google Scholar 

  • Zhao BL (2013) Differentiation of species of Bupleurum and its changes. Zhonghua Yi Shi Za Zhi 43(4):200–205

    PubMed  Google Scholar 

  • Zhou XT, Yang G, Zhou LF, Shan AJ, Wei Y, Chi XL, Li XL (2018) Study on characteristics and microscopic identification of fruits of Bupleurum chinense and Bupleurum Scorzonerifolium. ZhongGuo Xian Dai Zhong Yao 20(10):79–82

    Google Scholar 

  • Zhu SR, Meng QL, Zhu YA, Wang XR (2016) Investigation of phylogenetic relationship among three species of Channa based on ITS sequence analysis. Agric Biotechnol 5(1):50–53

    CAS  Google Scholar 

  • Zou XB (2011) Investigation on the application varieties of Bupleurum in the market. AnMo Yu KangFu Yi Xue 2(30):246–246

    Google Scholar 

Download references

Acknowledgements

The authors want to thank Shandong Baiweitang Chinese Herbal Medicine Drinks Slice CO., Ltd, SiChuan DePeiYuan Traditional Chinese Medicine Science & Technology Development Co., Ltd and Gansu Academy of Agricultural Sciences and Chinese Herbal Medicine Research Institute to provide Bupleurum L. seeds.The work was supported by Beijing Capital Medical Research Projiect (17ZY06).

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Authors and Affiliations

  1. School of Traditional Chinese Medicine, Capital Medical University, 10 You’anmen Outer West 1st Street, Beijing, 100069, China

    Wentao Qi, Julaiti Tuerxun, Jianchao Li, Chen Wang, Rong Luo & Changli Liu

  2. Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Beijing, 100069, China

    Rong Luo & Changli Liu

Authors
  1. Wentao Qi
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  2. Julaiti Tuerxun
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  3. Jianchao Li
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  4. Chen Wang
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  5. Rong Luo
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  6. Changli Liu
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Corresponding author

Correspondence to Changli Liu.

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Appendix

Appendix

See Figs. 6, 7, 8, 9, 10, 11 and 12.

Fig. 6
figure 6

A photograph showing Bupleurum seeds (Table 1). The photographs were taken under a stereomicroscope (SteREO Discovery.V8, Zeiss, Germany)

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Fig. 7
figure 7

DNA extraction and ITS-PCR amplification. a DNA extraction; b ITS-PCR product; a-M is 5 K DNA marker (300, 500, 800, 1000, 1500, 2000, 3000, 5000 bp); b-M is 2 K PlusIIDNA Marker (100, 250, 500, 750, 1000, 2000 bp etc.); 8–36 are the codes of the seeds

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Fig. 8
figure 8

Optimization of PCR amplification conditions for BCF/R8. M-2KPlusIIDNA Marker (100, 250, 500, 750, 1000, 2000 bp etc.); Bc-B.chinense; Bm-B. marginatum var.stenophyllum; Bs-B. scorzonerifolium; Bp-B. smithii var. parvifolium; Bf-B. falcatum; a DNA polymerase species: 2 × EasyTaq PCR SuperMix, 2 × Kapa Hifi HotStart ReadyMix, TaKaRa rTaq DNA polymerase; b annealing temperature: 52 °C, 55 °C, 58 °C and 61 °C; c cycle numbers:21, 22, 23 and 24 cycles; d DNA template: 0.5, 1.0, 1.5 and 2.0 µL (33 ng/µL); e PCR instruments: Veriti Dx PCR (Applied Biosystems), T100 PCR (Bole, USA), C1000 Touch PCR (Bole, USA)

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Fig. 9
figure 9

Optimization of PCR amplification conditions for BSF/R11. M-2KPlusIIDNA Marker (100, 250, 500, 750, 1000, 2000 bp etc.); Bc-B.chinense; Bm-B. marginatum var.stenophyllum; Bs-B. scorzonerifolium; Bp-B. smithii var. parvifolium; Bf-B. falcatum; a DNA polymerase species: 2 × EasyTaq PCR SuperMix, 2 × Kapa Hifi HotStart ReadyMix, TaKaRa rTaq DNA polymerase; b Annealing temperature: 45 °C, 48 °C, 50 °C and 55 °C; c cycle numbers:20, 25, 30 and 35 cycles; d DNA template: 0.5, 1.0, 1.5 and 2.0 µL (34 ng/µL); e PCR instruments: Veriti Dx PCR (Applied Biosystems), T100 PCR (Bole, USA), C1000 Touch PCR (Bole, USA)

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Fig. 10
figure 10

Optimization of PCR amplification conditions for BMSF/R1. M-2KPlusIIDNA Marker (100, 250, 500, 750, 1000, 2000 bp etc.); Bc-B.chinense; Bm-B. marginatum var.stenophyllum; Bs-B. scorzonerifolium; Bp-B. smithii var. parvifolium; Bf-B. falcatum; a DNA polymerase species: 2 × EasyTaq PCR SuperMix, 2 × Kapa Hifi HotStart ReadyMix, TaKaRa rTaq DNA polymerase; b Annealing temperature: 48 °C, 50 °C, 52 °C and 55 °C; c cycle numbers: 20, 25, 30 and 35 cycles; d DNA template: 0.5, 1.0, 1.5 and 2.0 µL (44 ng/µL); e PCR instruments: Veriti Dx PCR (Applied Biosystems), T100 PCR (Bole, USA), C1000 Touch PCR (Bole, USA)

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Fig. 11
figure 11

Optimization of PCR amplification conditions for BFF/R7. M-2KPlusIIDNA Marker (100, 250, 500, 750, 1000, 2000 bp etc.); Bc-B.chinense; Bm-B. marginatum var.stenophyllum; Bs-B. scorzonerifolium; Bp-B. smithii var. parvifolium; Bf-B. falcatum; a DNA polymerase species: 2 × EasyTaq PCR SuperMix, 2 × Kapa Hifi HotStart ReadyMix, TaKaRa rTaq DNA polymerase; b Annealing temperature: 48 °C, 50 °C, 52 °C and 55 °C; c cycle numbers:20, 21, 25, 30 and 35 cycles; d DNA template: 0.5, 1.0, 1.5 and 2.0 µL (43 ng/µL); e PCR instruments: Veriti Dx PCR (Applied Biosystems), T100 PCR (Bole, USA), C1000 Touch PCR (Bole, USA)

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Fig. 12
figure 12

Optimization of PCR amplification conditions for BSPF/R1. M-2KPlusIIDNA Marker (100, 250, 500, 750, 1000, 2000 bp etc.); Bc-B.chinense; Bm-B. marginatum var.stenophyllum; Bs-B. scorzonerifolium; Bp-B. smithii var. parvifolium; Bf-B. falcatum; a DNA polymerase species: 2 × EasyTaq PCR SuperMix, 2 × Kapa Hifi HotStart ReadyMix, TaKaRa rTaq DNA polymerase; b annealing temperature: 48 °C, 50 °C, 55 °C and 60 °C; c cycle numbers:20, 25, 30 and 35 cycles; d DNA template: 0.5, 1.0, 1.5 and 2.0 µL (30 ng/µL); e PCR instruments: Veriti Dx PCR (Applied Biosystems), T100 PCR (Bole, USA), C1000 Touch PCR (Bole, USA)

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Qi, W., Tuerxun, J., Li, J. et al. Identification of Bupleurum (Apiaceae) seeds by allele-specific PCR based on ITS sequences. 3 Biotech 10, 240 (2020). https://doi.org/10.1007/s13205-020-02233-1

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