Skip to main content
SpringerLink
Log in

Development of a new set of genic SSR markers in the genus Gentiana: in silico mining, characterization and validation

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

Abstract

Gentiana is an important genus of around 360 medicinally important species, majority of which are not well characterized. Despite its importance, very few genomic resources are available for Gentiana L. Till date, the number of informative and robust simple sequence repeat (SSR)-based markers is limited and very few efforts have been made for their development. A set of robust, freely accessible and informative SSR markers for Gentiana is a pre-requisite for any molecular systematic as well as improvement studies in this group of pharmacologically valuable plants. In view of the importance of these plants, Expressed Sequence Tag (EST) sequences of 18 Gentiana species were surveyed for the development of a large set of non-redundant SSR markers. A total of 5808 perfect SSR with an average length of 17 bp and relative abundance of 214 loci/Mb were identified in the analysed 47,487 EST sequences using Krait software. Mapping of the ESTs resulted in gene ontology annotations of 49.14% of the sequences. Based on these perfect SSRs, 2902 primer pairs were designed, and 60 markers were randomly selected and validated on a set of Gentiana kurroo Royle accessions. Among the screened markers, 39 (65%) were found to be cross-species transferable. This is the first report of the largest set of functional, novel genic SSR markers in Gentiana, which will be a valuable resource for future characterization, genotype identification, conservation and genomic studies in the various species of this group of important medicinal plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Aggarwal RK, Hendre PS, Varshney RK, Bhat PR, Krishnakumar V, Singh L (2007) Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species. Theor Appl Genet 114(2):359–372

    Article  PubMed  CAS  Google Scholar 

  • Anderson JA, Churchill GA, Autrique JE, Tanksley SD, Sorrells ME (1993) Optimizing parental selection for genetic linkage maps. Genome 36:181–186

    Article  PubMed  CAS  Google Scholar 

  • Behera MC, Raina R (2012) Gentiana kurroo Royale—a critically endangered medicinal herb. Int J Med Arom Plants 2(1):22–29

    Google Scholar 

  • Biswas MK, Bagchi M, Biswas D, Harikrishna JA, Liu Y, Li C, Sheng O, Mayer C, Yi G, Den G (2020) Genome-wide novel genic microsatellite marker resource development and validation for genetic diversity and population structure analysis of banana. Genes 11:1479

    Article  PubMed Central  CAS  Google Scholar 

  • Biswas MK, Xu Q, Mayer C, Deng X (2014) Genome wide characterization of short tandem repeat markers in sweet orange (Citrus sinensis). PLoS ONE 9(8):e104182

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Biswas MK, Liu Y, Li C, Sheng O, Mayer C, Yi G (2015) Genome-wide computational analysis of Musa microsatellites: classification, cross-taxon transferability, functional annotation, association with transposons & miRNAs, and genetic marker potential. PLoS ONE 10(6):e0131312

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cardle L, Ramsay L, Milbourne D, Macaulay M, Marshall D, Waugh R (2000) Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics 156(2):847–854

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chen C, Zhou P, Choi YA, Huang S, Gmitter FG (2006) Mining and characterizing microsatellites from citrus ESTs. Theor Appl Genet 112(7):1248–1257

    Article  PubMed  CAS  Google Scholar 

  • Davierwala AP, Reddy APK, Lagu MD, Ranjekar PK, Gupta VS (2001) Marker assisted selection of bacterial blight resistance genes in rice. Biochem Genet 39(7–8):261–278

    Article  PubMed  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure from small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Du L, Zhang C, Liu Q, Zhang X, Yue B (2018) Krait: an ultrafast tool for genome-wide survey of microsatellites and primer design. Bioinformatics 34(4):681–683

    Article  PubMed  CAS  Google Scholar 

  • Ellegren H (2004) Microsatellites: simple sequences with complex evolution. Nat Rev Genet 5(6):435–445

    Article  PubMed  CAS  Google Scholar 

  • Ellis J, Burke J (2007) EST-SSRs as a resource for population genetic analyses. Heredity 99:125–132

    Article  PubMed  CAS  Google Scholar 

  • Feng SP, Li WG, Huang HS, Wang JY, Wu YT (2009) Development, characterization and cross-species/genera transferability of EST-SSR markers for rubber tree (Hevea brasiliensis). Mol Breed 23(1):85–97

    Article  CAS  Google Scholar 

  • Filiz E, Koc I (2012) In silico chloroplast SSRs mining of Olea species. Biodiversitas 13:114–117

    Article  Google Scholar 

  • Folta KM, Staton M, Stewart PJ et al (2005) Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria × ananassa). BMC Plant Biol 5:12

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Gotz S, García-Gómez JM, Terol J et al (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36(10):3420–3435

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Gupta P, Varshney R (2000) The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica 113:163–185

    Article  CAS  Google Scholar 

  • Gupta S, Mukherjee A, Mondal M (2012) A census of Gentiana L. in India, systematics of flowering plants. In: Maiti G, Mukherjee SK (eds) Multidisciplinary approaches in angiosperm systematics, vol 1. University of Kalyani, Kalyani, pp 53–58

    Google Scholar 

  • Hendre PS, Aggarwal RK (2014) Development of genic and genomic SSR markers of robusta coffee (Coffea canephora Pierre Ex A. Froehner). PLoS One. 9(12):e113661

  • Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Izzah NK, Lee J, Jayakodi M, Perumal S, Jin M, Beom-Seok P, Ahn K, Yang TJ (2014) Transcriptome sequencing of two parental lines of cabbage (Brassica oleracea L. var capitata L.) and construction of an EST-based genetic map. BMC Genomics 15:149

    Article  PubMed  PubMed Central  Google Scholar 

  • Kanehisa FM, Tanabe M, Sato Y, Morishima K (2017) KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 45:D353–D361

    Article  PubMed  CAS  Google Scholar 

  • Kantety RV, Rota ML, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 4(5–6):501–510

    Article  Google Scholar 

  • Kohlein F (1991) Gentians. Christopher Helm, London

    Google Scholar 

  • La Rota L, Mauricio Kantety RV, Yu JK, Sorrells ME (2005) Non random distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley. BMC Genomics 6(1):23

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Li Y, Li LF, Chen GQ, Ge XJ (2007) Development of ten microsatellite loci for Gentiana crassicaulis (Gentianaceae). Conserv Genet 8:1499–1501

    Article  CAS  Google Scholar 

  • Masoudi-Nejad A, Tonomura K, Kawashima S et al (2006) EGassembler: online bioinformatics service for large-scale processing, clustering and assembling ESTs and genomic DNA fragments. Nucleic Acids Res 34:W459–W462

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Min XJ, Butler G, Storms R, Tsang A (2005) OrfPredictor: predicting protein-coding regions in EST-derived sequences. Nucleic Acids Res 33:W677–W680

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nat Genet 30(2):194–200

    Article  PubMed  CAS  Google Scholar 

  • Nakatsuka T, Yamada E, Saito M, Hikage T, Ushiku Y, Nishihara M (2012) Construction of the first genetic linkage map of Japanese gentian (Gentianaceae). BMC Genomics 13:672

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Nishihara M, Nakatsuka T, Mizutani-Fukuchi M, Tanaka Y, Yamamura S (2008) Gentians: from gene cloning to molecular breeding. Floric Ornam Biotech 5:57–67

    Google Scholar 

  • Pandey G, Misra G, Kumari K, Gupta S, Parida SK, Chattopadhyay D, Prasad M (2013) Genome-wide development and use of microsatellite markers for large-scale genotyping applications in foxtail millet (Setaria italica (L.)). DNA Res 20(2):197–207

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sardar SS, Pradhan K, Shukla RP, Saraswat R, Srivastava A, Jena SN, Das AB (2016) In silico mining of EST-SSRs in Arachis hypogaea L. and their utilization for genetic structure and diversity analysis in cultivars/breeding lines in Odisha India. Mol Breed 36:49

    Article  CAS  Google Scholar 

  • Sathishkumar R, Lakshmi PT, Annamalai A, Arunachalam V (2011) Mining of simple sequence repeats in the Genome of Gentianaceae. Pharmacognosy Res 3(1):19–29

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sato-Ushiku Y, Shimada N, Saito M, Yamada E, Hikage T, Nakatsuka T, Nishihara M (2011) Development of simple sequence repeat markers for identification of Japanese gentian cultivars. J Japan Soc Hort Sci 80:475–485

    Article  CAS  Google Scholar 

  • Shahi VK (1993) A significant drug of ayurveda: Vangamaya-katuki. Sachitra Ayurveda 46(1):21–27

    Google Scholar 

  • Sharma N, Chandel KPS, Paul A (1993) In vitro propagation of Gentiana kurroo—an indigenous threatened plant of medicinal importance. Plant Cell Tiss Organ Cult 34:3307–3309

    Article  Google Scholar 

  • Sharma R, Bhardwaj P, Negi R, Mohapatra T, Ahuja P (2009) Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.). BMC Plant Biol 9(1):53

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sharma N, Gowthami R, Devi SV, Malhotra EV, Pandey R, Agrawal A (2020) Cryopreservation of shoot tips of Gentiana kurroo Royle—a critically endangered medicinal plant of India. Plant Cell Tiss Organ Cult. https://doi.org/10.1007/s11240-020-01879-2

    Article  Google Scholar 

  • Singh A (2008) Phytochemicals of Gentianaceae: a review of pharmacological properties. Int J Pharm Sci Nanotech 1(1):33–36

    CAS  Google Scholar 

  • Singh BK, Mishra DC, Yadav S, Ambawat S, Vaidya E, Tribhuvan KU, Kumar A, Kumar S, Kumar S, Chaturvedi KK, Rani R, Yadav P, Rai A, Rai PK, Singh VV, Singh D (2016) Identification, characterization, validation and cross-species amplification of genic-SSRs in Indian mustard (Brassica juncea). J Plant Biochem Biotechnol 25:410–420

    Article  CAS  Google Scholar 

  • Skinder BM, Ganai BA, Wani AH (2017) Scientific study of Gentiana kurroo Royle. Medicines (basel, Switzerland) 4(4):74

    Google Scholar 

  • Sonah H, Deshmukh RK, Sharma A, Singh VP, Gupta DK, Gacche RN, Rana JC, Singh NK, Sharma TR (2011) Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium. PLoS ONE 6(6):e21298

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sorkheh K, Prudencio AS, Ghebinejad A, Dehkordi MK, Erogul D, Rubio M, Martínez-Gómez P (2016) In silico search, characterization and validation of new EST-SSR markers in the genus Prunus. BMC Res Notes 9:336

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Struwe L, Albert A (2002) Gentianaceae: systematic and natural history. Cambridge University Press, Cambridge, UK

    Google Scholar 

  • Sun SS, Fu PC, Cheng YW, Zhou XJ, Han JM (2018) Characterization and transferability of microsatellites for Gentiana lawrencei var. farreri (Gentianaceae). Appl Plant Sci 6(1):e1015

    Article  PubMed  PubMed Central  Google Scholar 

  • Sunita A, Bhattaracharya UC (1982) Some nomencultural notes on family Gentianaceae. Bull Bot Surv India 22:184–185

    Google Scholar 

  • Tang W, Eisenbrand G (1992) Gentiana spp. Chinese drugs of plant origin. Springer, Berlin, Heidelberg, pp 549–553

    Chapter  Google Scholar 

  • Thiel T, Michalek W, Varshney R, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 106(3):411–422

    Article  PubMed  CAS  Google Scholar 

  • Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3-new capabilities and interfaces. Nucleic Acids Res 40(15):e115

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Varshney RK, Graner A, Sorrells ME (2005a) Genic microsatellite markers in plants: features and applications. Trends Biotechnol 23:48–55

    Article  PubMed  CAS  Google Scholar 

  • Varshney RK, Sigmund R, Borner A, Korzun V, Stein N, Sorrells ME, Longridge P, Graner A (2005b) Interspecific transferability and comparative mapping of barley EST-SSR markers in wheat, rye and rice. Plant Sci 168:195–202

    Article  CAS  Google Scholar 

  • Ved D, Saha D, Ravikumar K, Haridasan K (2015) Gentiana kurroo. The IUCN Red List of Threatened Species 2015: e.T50126594A50131345. https://doi.org/10.2305/IUCN.UK.2015-2.RLTS.T50126594A50131345.en

  • Victoria FC, da Maia LC, de Oliveira AC (2011) In silico comparative analysis of SSR markers in plants. BMC Plant Biol 11:15

    Article  PubMed  PubMed Central  Google Scholar 

  • Yu JK, Dake TM, Singh S, Benscher D, Li W, Gill B, Sorrells ME (2004) Development and mapping of EST-derived simple sequence repeat (SSR) markers for hexaploid wheat. Genome 47:805–818

    Article  PubMed  CAS  Google Scholar 

  • Yumurtaci A, Sipahi H, Al-Abdallat A, Jighly A, Baum M (2017) Construction of new EST-SSRs for Fusarium resistant wheat breeding. Comput Biol Chem 68:22–28

    Article  PubMed  CAS  Google Scholar 

  • Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellite isolation: a review. Mol Ecol 11:1–16

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the Indian Council of Agricultural Research (ICAR) and Director, ICAR-NBPGR, New Delhi, India, for providing the funding and research facilities under an in-house project, for carrying out this work.

Author information

Authors and Affiliations

  1. Tissue Culture and Cryopreservation Unit, ICAR-National Bureau of Plant Genetic Resources, New Delhi, India

    Era Vaidya Malhotra, Rishu Jain, Sangita Bansal, Suresh Chand Mali, Neelam Sharma & Anuradha Agrawal

Authors
  1. Era Vaidya Malhotra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rishu Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sangita Bansal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Suresh Chand Mali
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Neelam Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anuradha Agrawal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Era Vaidya Malhotra.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary Information

Below is the link to the electronic supplementary material.

13205_2021_2969_MOESM1_ESM.pptx

Supplementary file1 Supplementary File 1: Gene ontology classification of EST-SSRs in G. macrophylla. ESTs grouped under (a) biological process (b) molecular function (c) cellular component. Supplementary File 2: Gene ontology classification of EST – SSRs in G. rigescens. ESTs grouped under (a) biological process (b) molecular function (c) cellular component. Supplementary File 3: Gene ontology classification of EST-SSRs in G. triflora. ESTs grouped under (a) biological process (b) molecular function (c) cellular component (PPTX 317 KB)

13205_2021_2969_MOESM2_ESM.xlsx

Supplementary file2 Supplementary File 4: List of total 2,902 EST-SSR markers designed for Gentiana species (XLSX 143 KB)

13205_2021_2969_MOESM3_ESM.docx

Supplementary file3 Supplementary File 5: Marker validation and cross-species transferability of Gentiana SSRs (DOCX 34 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Malhotra, E.V., Jain, R., Bansal, S. et al. Development of a new set of genic SSR markers in the genus Gentiana: in silico mining, characterization and validation. 3 Biotech 11, 430 (2021). https://doi.org/10.1007/s13205-021-02969-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-021-02969-4

Keywords

Search

Navigation