Article | . 2018 Vol. 36, Issue. 6
Authentication of Brassicae Semen and Brassicae Campestris Semen by DNA Barcode Analysis

Herbal Medicine Research Division, Korea Institute of Oriental Medicine1

2018.. 885:898


Brassica juncea and Brassica napus are economically important crops used for oilseed and forage who’s seed is also used for herbal medicines, namely Brassicae Semen and Brassicae Campestris Semen, respectively. Morphological discrimination of these two herbal medicines is difficult because the seeds of the Brassica species have similar shapes. Since the descriptions of the different Brassicae Semen in the Korean and Chinese pharmacopoeia can be confused, it is necessary to develop an accurate method for identification of the different species. Therefore, DNA barcodes of six Brassica species, B. juncea, B. napus, B. nigra, B. oleracea, B. rapa, and Sinapis alba, collected from various locations and markets, were analyzed to develop a molecular marker for distinguishing Brassicae Semen and Brassicae Campestris Semen. Nucleotide sequence comparison and phylogenetic analysis of internal transcribed spacer (ITS) regions based on the amphidiploid types and U's triangle provide useful genetic information for the identification of these two herbal medicines and Brassica species. However, the sequences of the chloroplast maturase K (matK) gene showed species-specific insertions and deletions or substitutions only for B. nigra, B. oleracea, and S. alba but not for B. juncea, B. napus, and B. rapa. The use of genome-specific marker nucleotides in ITS regions provide an objective and accurate method for authentication of Brassica species and valuable information for further molecular marker development.

1. Al-Shehbaz IA, Beilstein MA, Kellogg EA (2006) Systematics and phylogeny of the Brassicaceae (Cruciferae): an overview. Plant Syst Evol 259:89-120. doi:10.1007/s00606-006-0415-z  

2. Arias T, Pires JC (2012) A fully resolved chloroplast phylogeny of the brassica crops and wild relatives (Brassicaceae: Brassiceae): Novel clades and potential taxonomic implications. Taxon 61:980-988  

3. CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Natl Acad Sci 106:12794-12797. doi:10.1073/pnas.0905845106  

4. Chen F, Liu H, Yao Q, Fang P (2016) Evolution of mustard (Brassica juncea Coss) subspecies in China: evidence from the chalcone synthase gene. Genet Mol Res: GMR 15. doi:10.4238/gmr.15028045  

5. Chen S, Wan Z, Nelson MN, Chauhan JS, Redden R, Burton WA, Lin P, Salisbury PA, Fu T, et al (2013) Evidence from genome-wide simple sequence repeat markers for a polyphyletic origin and secondary centers of genetic diversity of Brassica juncea in China and India. J Hered 104:416-427. doi:10.1093/jhered/est015  

6. Cheng F, Wu J, Wang X (2014) Genome triplication drove the diversification of Brassica plants. Hortic Res 1:14024. doi:10.1038/hortres.2014.24  

7. Farris JS, Källersjö M, Kluge AG, Bult C (1994) Testing significance of incongruence. Cladistics 10:315-319. doi:10.1111/j.1096-0031.1994.tb00181.x  

8. Ghahramanzadeh R, Esselink G, Kodde L, Duistermaat H, Valkenburg J, Marashi S, Smulders M, Wiel C (2013) Efficient distinction of invasive aquatic plant species from non‐invasive related species using DNA barcoding. Mol Ecol Res 13:21-31. doi:10.1111/1755-0998.12020  

9. Hebert PD, Ratnasingham S, de Waard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Lond B Biol Sci 270:S96-S99. doi:10.1098/rsbl.2003.0025  

10. Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS One 6:e19254. doi:10.1371/journal.pone.0019254  

11. Järvinen P, Palmé A, Morales LO, Lännenpää M, Keinänen M, Sopanen T, Lascoux M (2004) Phylogenetic relationships of Betula species (Betulaceae) based on nuclear ADH and chloroplast matK sequences. Am J Bot 91:1834-1845. doi:10.3732/ajb.91.11.1834  

12. Johnston JS, Pepper AE, Hall AE, Chen ZJ, Hodnett G, Drabek J, Lopez R, Price HJ (2005) Evolution of genome size in Brassicaceae. Ann Bot 95:229-235. doi:10.1093/aob/mci016  

13. Khan MA, Rabbani MA, Munir M, Ajmal SK, Malik MA (2008) Assessment of genetic variation within Indian mustard (Brassica juncea) germplasm using random amplified polymorphic DNA markers. J Integr Plant Biol 50:385-392. doi:10.1111/j.1744-7909.2007.00630.x  

14. Kim W, Ji Y, Choi G, Kang Y, Yang S, Moon B (2016) Molecular identification and phylogenetic analysis of important medicinal plant species in genus Paeonia based on rDNA-ITS, matK, and rbcL DNA barcode sequences. Genet Mol Res: GMR 15. doi:10.4238/gmr.15038472  

15. Korea Food and Drug Administration (2012) The Korean Herbal Pharmacophoeia 4th edition. 1st supplement, 2013. Partial amendment, 2016, 2017. The Korea Food and Drug Administration, Seoul, Korea, pp 8-9  

16. Korea Institute of Oriental Medicine (2018) Defining dictionary for medicinal herbs [Korean, ‘Hanyak Giwon Sajeon’]. Published on the Internet; Accessed 7 February 2018  

17. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870-1874. doi:10.1093/molbev/msw054  

18. Kwon S, Lim K, Lim M, Park J, Kim J, Kim J, Lee S, Park B, Jin Y (2007) Molecular genetics of Brassicaceae based on AFLP display. Korean J Hortic Sci Technol 25:75-81  

19. Lee J, Kim CS, Lee IY (2015) Molecular identification of Pooideae, Poaceae in Korea. Weed Turf Sci 4:18-25. doi:10.5660/WTS.2015.4.1.18  

20. Li P, Zhang S, Li F, Zhang S, Zhang H, Wang X, Sun R, Bonnema G, Borm TJ (2017) A phylogenetic analysis of chloroplast genomes elucidates the relationships of the six economically important Brassica species comprising the Triangle of U. Front Plant Sci 8:111. doi:10.3389/fpls.2017.00111  

21. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451-1452. doi:10.1093/bioinformatics/btp187  

22. Lim Y, Yook MJ, Zhang CJ, Nah G, Park S, Kim DS (2015) Dormancy associated weedy risk of the F1 hybrid resulted from gene flow from oilseed rape to mustard. Weed Turf Sci 4:35-43. doi:10.5660/WTS.2015.4.1.35  

23. Lü N, Yamane K, Ohnishi O (2008) Genetic diversity of cultivated and wild radish and phylogenetic relationships among Raphanus and Brassica species revealed by the analysis of trnK/matK sequence. Breed Sci 58:15-22. doi:10.1270/jsbbs.58.15  

24. Moon B, Kim W, Ji Y, Lee Y, Kang Y, Choi G (2016) Molecular identification of the traditional herbal medicines, Arisaematis Rhizoma and Pinelliae Tuber, and common adulterants via universal DNA barcode sequences. Genet Mol Res 15(1):gmr.15017064. doi:10.4238/ gmr.15017064  

25. Moon B, Park I, Kim W, Yang S, Kang Y (2018) The complete chloroplast genome sequence of Fritillaria thunbergii Miq., an important medicinal plant, and identification of DNA markers to authenticate Fritillariae Bulbus. Hortic. Environ. Biotechnol 59:71-80. doi:10.1007/s13580-018-0008-0  

26. Nagaharu U (1935) Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Jpn J Bot 7:389-452  

27. Nilsson RH, Tedersoo L, Abarenkov K, Ryberg M, Kristiansson E, Hartmann M, Schoch CL, Nylander JA, Bergsten J, et al (2012) Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences. MycoKeys 4:37. doi:10.3897/ mycokeys.4.3606  

28. Ramchiary N, Nguyen VD, Li X, Hong CP, Dhandapani V, Choi SR, Yu G, Piao ZY, Lim YP (2011) Genic microsatellite markers in Brassica rapa: development, characterization, mapping, and their utility in other cultivated and wild Brassica relatives. DNA Res 18:305-320. doi:10.1093/dnares/dsr017  

29. Srivastava A, Gupta V, Pental D, Pradhan A (2001) AFLP-based genetic diversity assessment amongst agronomically important natural and some newly synthesized lines of Brassica juncea. Theor Appl Genet 102:193-199. doi:10.1007/s001220051635  

30. Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4.0. Sinauer Associates, Sunderland, Massachusetts, USA  

31. Warwick SI, Francis A, Al-Shehbaz IA (2006) Brassicaceae: species checklist and database on CD-Rom. Plant Syst Evol 259:249-258. doi:10.1007/s00606-006-0421-1  

32. Warwick SI, Sauder CA (2005) Phylogeny of tribe Brassiceae (Brassicaceae) based on chloroplast restriction site polymorphisms and nuclear ribosomal internal transcribed spacer and chloroplast trn L intron sequences. Can J Bot 83:467-483. doi:10.1139/b05-021  

33. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. Academic Press, Inc., NY, USA, pp 315-322. doi:10.1016/B978-0-12-372180-8.50042-1  

34. Yang J, Liu D, Wang X, Ji C, Cheng F, Liu B, Hu Z, Chen S, Pental D, et al (2016a) The genome sequence of allopolyploid Brassica juncea and analysis of differential homoeolog gene expression influencing selection. Nat Genet 48:1225-1232. doi:10.1038/ng.3657  

35. Yang K, Yi G, Arif Hasan Khan R, Jeong N, Lee Y, Park J, Kim H, Chung M, Nou I (2016b) Development of molecular markers for varietal identification of Brassica juncea on the basis of the polymorphic sequence of ITS regions and MITE families. Korean J Hortic Sci Technol 34:305-313  

36. Zhang Z-L, Song M-F, Guan Y-H, Li H-T, Niu Y-F, Zhang L-X, Ma X-J (2015) DNA barcoding in medicinal plants: testing the potential of a proposed barcoding marker for identification of Uncaria species from China. Biochem Syst Ecol 60:8-14. doi:10.1016/j.bse.2015.02.017