SSR marker development in Clerodendrum trichotomum using transcriptome sequencing

Autoři: Gongwei Chen aff001;  Yuanzheng Yue aff001;  Yajie Hua aff001;  Die Hu aff001;  Tingting Shi aff001;  Zhaojing Chang aff001;  Xiulian Yang aff001;  Lianggui Wang aff001
Působiště autorů: College of Landscape Architecture, Nanjing Forestry University, Nanjing, Jiangsu, China aff001;  College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu, China aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225451


Clerodendrum trichotomum, a member of the Lamiaceae (Verbenaceae) family, is an ornamental plant widely distributed in South Asia. Previous studies have focused primarily on its growth characteristics, stress resistance, and pharmacological applications; however, molecular investigations remain limited. Considering germplasm conservation and the extensive applications of this plant, it is necessary to explore transcriptome resources and SSR makers for C. trichotomum. In the present study, RNA sequencing was used to determine the transcriptome of C. trichotomum. Subsequently, unigene annotations and classifications were obtained, and SSRs were mined with MIcroSAtellite. Finally, primer pairs designed with Oligo 6.0 were selected for polymorphism validation. In total, 127,325,666 high-quality reads were obtained, and 58,345 non-redundant unigenes were generated, of which 36,900 (63.24%) were annotated. Among the annotated unigenes, 35,980 (97.51%) had significant similarity to 607 species in Nr databases. In addition, a total of 6,444 SSRs were identified in 5,530 unigenes, and 200 random primer pairs were designed for polymorphism validation. Furthermore, after primary polymorphism identification, 30 polymorphic primer pairs were selected for the further polymorphism screening, and 200 alleles were identified, 197 of which showed polymorphism. In this work, a large number of unigenes were generated, and numerous SSRs were detected. These findings should be beneficial for further investigations into germplasm conservation and various applications of C. trichotomum. These results should also provide a solid foundation for future molecular biology studies in C. trichotomum.

Klíčová slova:

Alleles – DNA – Microsatellite loci – Plant breeding – RNA sequencing – Sequence motif analysis – Shannon index – Transcriptome analysis


1. Xu RL, Wang R, Ha W, Shi YP. New cyclohexylethanoids from the leaves of Clerodendrum trichotomum. Phytochemistry Letters. 2014; 7: 111–113. doi: 10.1016/j.phytol.2013.10.010

2. Xu RL, Jiang H, Wang R, Shi YP. Diverse Terpenoids from the Leaves of Clerodendrum trichotomum. Chemistry of Natural Compounds. 2015; 51(5): 999–1000.

3. Sakamoto RL, Motomi I, Nobumitsu K. Contribution of Pollinators to Seed Production as Revealed by Differential Pollinator Exclusion in Clerodendrum trichotomum (Lamiaceae). PLOS ONE. 2012; 7(3): e33803. doi: 10.1371/journal.pone.0033803 22442724

4. Govindarajan M, Rajeswary M, Hoti SL, Murugan K, Kovendan K, Arivoli S, et al. Clerodendrum chinense-mediated biofabrication of silver nanoparticles: Mosquitocidal potential and acute toxicity against non-target aquatic organisms. Journal of Asia-Pacific Entomology. 2015; 19(1): 51–58. doi: 10.1016/j.aspen.2015.11.009

5. Tian J, Zhao QS, Zhang HJ, Lin ZW, Sun HD. New Cleroindicins from Clerodendrum indicum. Journal of Natural Products. 1997; 60: 766–769. doi: 10.1021/np9606759

6. Miyake T, Inoue K. Character displacement in style length between pollinator-sharing Clerodendrum trichotomum and C. izuinsulare (Verbenaceae). Plant Systematics and Evolution. 2003; 243(1–2): 31–38.

7. Thitilertdecha P, Guy RH, Rowan MG. Characterisation of polyphenolic compounds in Clerodendrum petasites S. Moore and their potential for topical delivery through the skin. Journal of Ethnopharmacology. 2014; 154(2): 400–407. doi: 10.1016/j.jep.2014.04.021 24747028

8. Muthu C, Reegan AD, Kingsley S, Ignacimuthu S. Larvicidal activity of pectolinaringenin from Clerodendrum phlomidis L. against Culex quinquefasciatus Say and Aedes aegypti L. (Diptera: Culicidae). Parasitology Research. 2012; 111(3): 1059–1065. doi: 10.1007/s00436-012-2932-8 22562213

9. Ji WC, Cho EJ, Dong GL, Choi K, Ku J, Park KW, et al. Antibacterial Activity of Triterpenoids from Clerodendron trichotomum. Journal of Applied Biological Chemistry. 2012; 55(3): 169–172.

10. Wang WX, Xiong J, Tang Y, Zhu JJ, Li M, Zhao Y, et al. Rearranged abietane diterpenoids from the roots of Clerodendrum trichotomum and their cytotoxicities against human tumor cells. Phytochemistry. 2013; 89(9): 89–95. doi: 10.1016/j.phytochem.2013.01.008 23462587

11. Li LZ, Wang MH, Sun JB, Liang JY. Abietane diterpenoids and other constituents from Clerodendrum trichotomum. Biochemical Systematics and Ecology. 2014; 56: 218–220. doi: 10.1016/j.bse.2014.06.002

12. Wang JH, Luan F, He XD, Wang Y, Li MX. Traditional uses and pharmacological properties of Clerodendrum phytochemicals. Journal of Traditional and Complementary Medicine. 2017; 8(1): 24–38. doi: 10.1016/j.jtcme.2017.04.001 29321986

13. Chae S, Kang KA, Kim JS, Kim HK, Lee EJ, Hyun JW, et al. Antioxidant activities of acetylmartynosides from Clerodendron trichotomum. Journal of Applied Biological Chemistry. 2007; 50(4): 270–274.

14. Wahba HM, Abouzid SF, Sleem AA, Apers S, Pieters L, Shahat AA. Chemical and biological investigation of some Clerodendrum species cultivated in Egypt. Pharmaceutical Biology. 2011; 49(1): 66–72. doi: 10.3109/13880209.2010.494674 20738216

15. Mizusawa L, Kaneko S, Hasegawa M, Isagi Y. Development of Nuclear SSRs for the insular shrub Clerodendrum izuinsulare(Verbenaceae) and the widespread C. trichotomum. American Journal of Botany. 2011; 98(11): e333–e336. doi: 10.3732/ajb.1100155 22025293

16. Kaur S, Pembleton LW, Cogan NO, Savin KW, Leonforte T, Paull J, et al. Transcriptome sequencing of field pea and faba bean for discovery and validation of SSR genetic markers. Bmc Genomics. 2012; 13(1): 104. doi: 10.1186/1471-2164-13-104 22433453

17. Zalapa JE, Cuevas H, Zhu H, Steffan S, Senalik D, Zeldin E, et al. Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences. American Journal of Botany. 2012; 99(2): 193–208. doi: 10.3732/ajb.1100394 22186186

18. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, et al. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Molecular Breeding. 1996; 2(3): 225–238. doi: 10.1007/bf00564200

19. Wang L, Yang X, Addisu G, Cui S, Guojun MU, Liu L, et al. Screening for Polymorphic Primer Pairs and Optimization of AFLP Marker System in Peanut. Journal of Nuclear Agricultural Sciences. 2017; 31(11): 2087–2095.

20. Singh RK, Bhatia VS, Bhat KV, Mohapatra T, Singh NK, Bansal KC, et al. SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity. Genetics and Molecular Biology. 2010; 33(2): 319–324. doi: 10.1590/S1415-47572010005000024 21637488

21. Ronoh R, Linde M, Winkelmann T, Abukutsa-Onyango M, Dinssa FF, Debener T. Development of next-generation sequencing (NGS)-based SSRs in African nightshades: Tools for analyzing genetic diversity for conservation and breeding. Scientia Horticulturae. 2018; 235: 152–159. doi: 10.1016/j.scienta.2018.03.003

22. Xu M, Liu X, Wang JW, Teng SY, Shi JQ, Li YY, et al. Transcriptome sequencing and development of novel genic SSR markers for Dendrobium officinale. Molecular Breeding. 2017; 37(2): 18. doi: 10.1007/s11032-016-0613-5

23. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology. 2011; 29(7): 644–652. doi: 10.1038/nbt.1883 21572440

24. Arokiyaraj S, Sripriya N, Bhagya R, Radhika B, Prameela L, Udayaprakash NK. Phytochemical screening, antibacterial and free radical scavenging effects of Artemisia nilagirica, Mimosa pudica and Clerodendrum siphonanthus-An in-vitro study. Asian Pacific Journal of Tropical Biomedicine. 2012; 2(2): 601–604. doi: 10.1016/S2221-1691(12)60281-0

25. Olmstead R G., Steane D A., Mabberley D J., Yuan YW. Further disintegration and redefinition of Clerodendrum (Lamiaceae): Implications for the understanding of the evolution of an intriguing breeding strategy. Taxon. 2010; 59(1): 125–133. doi: 10.2307/27757057

26. Carver M, Blüthgen N, Grimshaw JF, Bellis GA. Aphis clerodendri Matsumura (Hemiptera: Aphididae), attendant ants (Hymenoptera: Formicidae) and associates on Clerodendrum (Verbenaceae) in Australia. Austral Entomology. 2014; 42(2): 109–113. doi: 10.1046/j.1440-6055.2003.00339.x

27. Ono M, Furusawa C, Matsumura K, Noguchi S, Yasuda S, Okawa M, et al. A new diterpenoid from the leaves of Clerodendron trichotomum. Journal of Natural Medicines. 2013; 67(2): 404–409. doi: 10.1007/s11418-012-0690-7 22825680

28. Zhang R, Zhu AD, Wang XJ, Yu J, Zhang HR, Gao JS, et al. Development of Juglans Regia SSR Markers by Data Mining of the EST Database. Plant Molecular Biology Reporter. 2010; 28(4): 646–653. doi: 10.1007/s11105-010-0192-2

29. Guo LN, Zhao XL, Gao XF. De novo assembly and characterization of leaf transcriptome for the development of EST-SSR markers of the non-model species Indigofera szechuensis. Biochemical Systematics and Ecology. 2016; 68: 36–43. doi: 10.1016/j.bse.2016.06.010

30. Huang HY. Development of SSR Molecular Markers Based on Transcriptome Sequencing of Eucommia ulmoides. Scientia Silvae Sinicae. 2013; 49(5): 176–181. doi: 10.11707/j.1001-7488.20130523

31. Chen C, Xu M, Wang C, Qiao G, Wang W, Tan Z, et al. Characterization of the Lycium barbarum fruit transcriptome and development of EST-SSR markers. PlOS ONE. 2017; 12(11): e0187738. doi: 10.1371/journal.pone.0187738 29125846

32. Soriano JM, Romero C, Vilanova S, Llácer G, Badenes ML. Genetic diversity of loquat germplasm (Eriobotrya japonica (Thunb) Lindl) assessed by SSR markers. Genome. 2005; 48(1): 108–114. doi: 10.1139/g04-101 15729402

33. Wei W, Qi X, Wang L, Zhang Y, Hua W, Li D, et al. Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers. BMC Genomics. 2011; 12: 451–462. doi: 10.1186/1471-2164-12-451 21929789

34. Meng D, Zhang T, Hu Y, Zhou H, Woeste K, Zhao P. De Novo Assembly and Characterization of Bud, Leaf and Flowers Transcriptome from Juglans Regia L. for the Identification and Characterization of New EST-SSRs. Forests. 2016; 7(10): 247–263. doi: 10.3390/f7100247

35. Li DJ, Deng Z, Qin B, Liu XH, Men ZH. De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.). Bmc Genomics. 2012; 13: 192. doi: 10.1186/1471-2164-13-192 22607098

36. Ying W, Liu K, Bi D, Zhou S, Shao J. Characterization of the transcriptome and EST-SSR development in Boea clarkeana, a desiccation-tolerant plant endemic to China. Peerj. 2017; 5(2): e3422. doi: 10.7717/peerj.3422 28630801

37. Pan L, Fu J, Zhang R, Qin Y, Lu F, Jia L, et al. Genetic diversity among germplasms of Pitaya based on SSR markers. Scientia Horticulturae. 2017; 225: 171–176. doi: 10.1016/j.scienta.2017.06.053

38. Niemandt M, Roodt-Wilding R, Tobutt KR, Bester C. Microsatellite marker applications in Cyclopia (Fabaceae) species. South African Journal of Botany. 2018; 116: 52–60. doi: 10.1016/j.sajb.2018.02.408

39. Yoder AD, Poelstra J, Tiley GP, Williams R. Neutral Theory is the Foundation of Conservation Genetics. Molecular Biology and Evolution. 2018; 35(6): 1322–1326. doi: 10.1093/molbev/msy076 29669008

40. Fan L, Zhang MY, Liu QZ, Li LT, Song Y, Wang LF, et al. Transferability of Newly Developed Pear SSR Markers to Other Rosaceae Species. Plant Mol Biol Report. 2018; 116: 52–60. doi: 10.1016/j.sajb.2018.02.408

41. Gang Q, Ping J, Wang D, Zhen Z, Luo S, Yang M. Malt Genotypic Screening of Polymorphism Information Content (PIC) of SSR Markers Based on Physiological Traits in Barley. Journal of Molecular Biology. 2011; 25(7): 66–77.

Článek vyšel v časopise


2019 Číslo 11