Genome-wide mining of microsatellites in king cobra (Ophiophagus hannah) and cross-species development of tetranucleotide SSR markers in Chinese cobra (Naja atra)

  • Wencong Liu
  • Yongtao Xu
  • Zekun Li
  • Jun Fan
  • Yi YangEmail author
Original Article


The complete genome sequence provides the opportunity for genome-wide and coding region analysis of SSRs in the king cobra and for cross-species identification of microsatellite markers in the Chinese cobra. In the Ophiophagus hannah genome, tetranucleotide repeats (38.03%) were the most abundant category, followed by dinucleotides (23.03%), pentanucleotides (13.07%), mononucleotides (11.78%), trinucleotides (11.49%) and hexanucleotides (2.6%). Twenty predominant motifs in the O. hannah genome were (A)n (C)n, (AC)n, (AG)n, (AT)n, (AGG)n, (AAT)n, (AAG)n, (AAC)n, (ATG)n, (ATAG)n, (AAGG)n, (ATCT)n, (CCTT)n, (ATTT)n, (AAAT)n, (AATAG)n, (ATTCT)n, (ATATGT)n, (AGATAT)n. In total, 4344 SSRs were found in coding sequences (CDSs). Tetranucleotides (52.79%) were the most abundant microsatellite type in CDS, followed by trinucleotides (28.50%), dinucleotides (11.02%), pentanucleotides (4.42%), mononucleotides (1.77%), and hexanucleotides (1.50%). A total of 984 CDSs containing microsatellites were assigned 11152 Gene Ontology (GO) functional terms. Gene Ontology (GO) analysis demonstrated that cellular process, cell and binding were the most frequent GO terms in biological process, cellular component and molecular function, respectively. Thirty-two novel highly polymorphic (PIC > 0.5) SSR markers for Naja atra were developed from cross-species amplification based on the tetranucleotide microsatellite sequences in the king cobra genome. The number of alleles (NA) per locus had between 3 and 11 alleles with an average of 6.5, the polymorphism information content (PIC) value ranged from 0.521 to 0.858 (average = 0.707), the observed heterozygosity (Ho) of 32 microsatellite loci ranged from 0.292 to 0.875 (mean = 0.678), the expected heterozygosity (HE) ranged from 0.561 to 0.889 (average = 0.761), and 3 microsatellite loci exhibited statistically significant departure from Hardy–Weinberg equilibrium (HWE) after Bonferroni correction (p < 0.003).


King cobra Chinese cobra Genome SSR markers Tetranucleotide repeats Polymorphic 



This research was funded by 973 Projects NSFC31671455 and 2015CB755702.

Compliance with ethical standards

Conflicts of interest

There are no conflicts of interest among authors.

Supplementary material

11033_2019_5044_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 17 kb)
11033_2019_5044_MOESM2_ESM.csv (2 mb)
Supplementary material 2 (CSV 2048 kb)
11033_2019_5044_MOESM3_ESM.xlsx (26 kb)
Supplementary material 3 (XLSX 26 kb)


  1. 1.
    Tautz D (1989) Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res 17(16):6463–6471Google Scholar
  2. 2.
    Powell W, Morgante M, Mcdevitt R, Vendramin GG, Rafalski JA (1995) Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines. Proc Natl Acad Sci USA 92(17):7759–77633Google Scholar
  3. 3.
    Sun J, University NA, Entomology DO (2012) The application of microsatellite markers in insect molecular ecology. J Nanjing Agric Univ 11(1):1–6Google Scholar
  4. 4.
    Li Y, Korol AB, Fahima T, Beiles A, Nevo E (2010) Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review. Mol Ecol 11(12):2453–2465Google Scholar
  5. 5.
    Wissler L, Godmann L, Bornbergbauer E (2012) Evolutionary dynamics of simple sequence repeats across long evolutionary time scale in genus Drosophila. Trends Evol Biol 4(1):34–44Google Scholar
  6. 6.
    Kim KS, Ratcliffe ST, French BW, Liu L, Sappington TW (2008) Utility of EST-derived SSRS as population genetics markers in a beetle. J Hered 99(2):112Google Scholar
  7. 7.
    Lópezsepúlveda P, Takayama K, Greimler J, Crawford DJ, Peñailillo P, Baeza M et al (2016) Speciation and biogeography of erigeron (Asteraceae) in the Juan Fernández Archipelago, Chile, based on AFLPS and SSRS. Syst Bot 40(3):888–899Google Scholar
  8. 8.
    Bao Y, Zhou HF, Hong DY, Ge S (2006) Genetic diversity and evolutionary relationships of oryza, species with the b- and c-genomes as revealed by SSR markers. J Plant Biol 49(5):339–347Google Scholar
  9. 9.
    Gaurav S, Pérez-Pulido AJ, Thac D, Seong TY, Casimiro-Soriguer CS, Nicola LP et al (2016) Plantfuncssr: integrating first and next generation transcriptomics for mining of SSR-functional domains markers. Front Plant Sci 7:878Google Scholar
  10. 10.
    Ditta A, Zhou Z, Cai X, Shehzad M, Wang X, Okubazghi K et al (2018) Genome-wide mining and characterization of SSR markers for gene mapping and gene diversity in Gossypium barbadense L. and Gossypium darwinii G. Watt accessions. Agronomy 8(9):181Google Scholar
  11. 11.
    Wang Q, Fang L, Chen J, Hu Y, Si Z, Wang S et al (2015) Genome-wide mining, characterization, and development of microsatellite markers in gossypium species. Sci Rep 5:10638Google Scholar
  12. 12.
    Saha D, Rana RS, Das S, Datta S, Mitra J, Cloutier SJ et al (2019) Genome-wide regulatory gene-derived SSRs reveal genetic differentiation and population structure in fiber flax genotypes. J Appl Genet 60(1):13–25Google Scholar
  13. 13.
    Xuewen W, Le W (2016) Gmata: an integrated software package for genome-scale SSR mining, marker development and viewing. Front Plant Sci 7:1350Google Scholar
  14. 14.
    Avvaru AK, Saxena S, Sowpati DT, Mishra RK (2017) MSDB: a comprehensive database of simple sequence repeats. Genome Biol Evol 9(6):1797–1802Google Scholar
  15. 15.
    Du L, Zhang C, Liu Q, Zhang X, Yue B (2017) Krait: an ultrafast tool for genome-wide survey of microsatellites and primer design. Bioinformatics 34(4):681–683Google Scholar
  16. 16.
    Tin M, Rai M, Maung C, Tun P, Warrell DA (1991) Bites by the King cobra (Ophiophagus hannah) in Myanmar: successful treatment of severe neurotoxic envenoming. Q J Med 80(293):751–762Google Scholar
  17. 17.
    Vonk FJ, Casewell NR, Henkel CV, Heimberg AM, Jansen HJ, Mccleary RJ et al (2013) The King cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. PNAS 110(51):20651–20656Google Scholar
  18. 18.
    Wang H, He H (2018) Characterization of multidrug-resistant Klebsiella pneumoniae isolated from the Chinese cobra Naja atra in a Beijing suburb. Biocell 42(2):47–54Google Scholar
  19. 19.
    Zhou Z, Jiang Z (2004) International trade status and crisis for snake species in China. Conserv Biol 18(5):1386–1394Google Scholar
  20. 20.
    Lin LH, Mao LX, Luo X, Qu YF, Ji X (2011) Isolation and characterization of microsatellite loci in the chinese cobra Naja atra (Elapidae). Int J Mol Sci 12(7):4435–4440Google Scholar
  21. 21.
    Yu D, Zheng XX, Yao YT, Lin LH (2015) Development of microsatellite markers in the rice paddy snake Enhydris plumbea, (Colubridae). Conserv Genet Resour 7(1):155–156Google Scholar
  22. 22.
    Bergey CM, Pozzi L, Disotell TR et al (2013) A new method for genome-wide marker development and genotyping holds great promise for molecular primatology. Int J Primatol 34(2):303–314Google Scholar
  23. 23.
    Zhang S, Tang C, Zhao Q, Li J, Yang L, Qie L et al (2014) Development of highly polymorphic simple sequence repeat markers using genome-wide microsatellite variant analysis in foxtail millet [Setaria italica (L.) P. Beauv]. BMC Genom 15(1):78Google Scholar
  24. 24.
    Xu Y, Hu Z, Wang C, Zhang X, Li J, Yue B (2016) Characterization of perfect microsatellite based on genome-wide and chromosome level in Rhesus monkey (Macaca mulatta). Gene 592(2):269–275Google Scholar
  25. 25.
    Altschul SF, Madden TL, Zhang J et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein detabase search programs. Nucleic Acids Res 25(17):3389–3402Google Scholar
  26. 26.
    Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M et al (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18):3674–3676Google Scholar
  27. 27.
    Ye J, Fang L, Zheng HK et al (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:293–297Google Scholar
  28. 28.
    Young MD, Wakefield MJ, Symth GK et al (2012) GOSEQ: gene ontology testing for RNA-seq datasets. R BioconductorGoogle Scholar
  29. 29.
    Maere S, Heymans K, Kuiper M (2005) Bingo: a cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21(16):3448–3449Google Scholar
  30. 30.
    Jones R, Cable J, Bruford MW (2008) An evaluation of non-invasive sampling for genetic analysis in northern European reptiles. Herpetol J 18(1):32–39Google Scholar
  31. 31.
    Li Q, Wan JM (2005) SSR hunter: development of a local searching software for SSR sites. Hereditas 27(5):808–810Google Scholar
  32. 32.
    Lalitha S (2000) Primer premier 5. Biotech Softw Internet Rep 1(6):270–272Google Scholar
  33. 33.
    Ju J, Ruan C, Fuller CW, Glazer AN et al (1995) Fluorescence energy transfer dye-labeled primers for dna sequencing and analysis. Proc Natl Acad Sci USA 92(10):4347–4351Google Scholar
  34. 34.
    Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18(2):233–234Google Scholar
  35. 35.
    Marshall TC, Slate J, Kruuk LEB et al (1998) Statistical confidence for likelihood-based paternity inference in natural populations. Mol Ecol 7(5):639–655Google Scholar
  36. 36.
    Raymond M, Rousset F (1995) Genepop (version 1.22): population genetics software for exact tests and ecumenicism. Heredity 86(3):248–249Google Scholar
  37. 37.
    Nguyen TTB, Rimatsu A, Hong Y et al (2015) Genome-wide characterization of microsatellites and marker development in the carcinogenic liver Flukeclonorchis sinensis. Parasitol Res 114(6):2263–2272Google Scholar
  38. 38.
    Ma ZJ (2015) Genome-wide characterization of perfect microsatellites in yak (Bos grunniens). Genetica 143(4):515–520Google Scholar
  39. 39.
    Liu S, Hou W, Sun T, Xu Y, Li P, Yue B et al (2017) Genome-wide mining and comparative analysis of microsatellites in three macaque species. Mol Genet Genomics 292(3):537–550Google Scholar
  40. 40.
    Todd AC, Alexander WP, Jason DE, Koning AP et al (2012) Rapid microsatellite identification from illumina paired-end genomic sequencing in two birds and a snake. PLoS ONE 7(2):e30953Google Scholar
  41. 41.
    Qian J, Xu H, Song J, Xu J, Zhu Y, Chen S (2013) Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum. Gene (Amsterdam) 512(2):331–336Google Scholar
  42. 42.
    Jessy LMC, Morin E, Tacon FL, Martin F (2011) Survey and analysis of simple sequence repeats in thelaccaria bicolorgenome, with development of microsatellite markers. Curr Genet 57(2):75–88Google Scholar
  43. 43.
    Siddle KJ, Goodship JA, Keavney B, Santibanez-Koref MF (2011) Bases adjacent to mononucleotide repeats show an increased single nucleotide polymorphism frequency in the human genome. Bioinformatics 27(7):895–898Google Scholar
  44. 44.
    Qi WH, Jiang XM, Du LM et al (2015) Genome-wide survey and analysis of microsatellite sequences in bovid species. PLoS ONE 10(7):e0133667Google Scholar
  45. 45.
    Vianna JA, Noll D, Murajornet I et al (2017) Comparative genome-wide polymorphic microsatellite markers in Antarctic penguins through next generation sequencing. Genet Mol Biol 40(3):676–687Google Scholar
  46. 46.
    Bai C, Liu S, Zhuang Z (2016) Characteristic analysis of microsatellite DNA in the genome of Gobiidae. Prog Fish Sci 05:9–15Google Scholar
  47. 47.
    Wang C, Du LM, Li P et al (2015) Distribution patterns of microsatellites in the genome of the German cockroach (Blattella germanica). Acta Entomol Sin 58(10):1037–1045Google Scholar
  48. 48.
    Mckinney K, Sprecher S, Orbuch TL (2003) Genome-wide analysis of microsatellite repeats in humans: their abundance and density in specific genomic regions. Genome Biol 4(2):1–10Google Scholar
  49. 49.
    Huang J, Li YZ, Du LM, Yang B, Shen FJ, Zhang HM et al (2015) Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system. BMC Genom 16(1):61Google Scholar
  50. 50.
    Castoe TA, Poole AW, Wanjun GU, Koning APJD, Daza JM, Smith EN et al (2010) Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence. Mol Ecol Resour 10(2):341–347Google Scholar
  51. 51.
    Glenn TC, Lance SL, Mckee AM, Webster BL, Faircloth BC (2013) Significant variance in genetic diversity among populations of schistosoma haematobium detected using microsatellite dna loci from a genome-wide database. Parasites Vectors 6(1):300Google Scholar
  52. 52.
    Castagnone-Sereno P, Danchin EG, Deleury E, Guillemaud T, Malausa T, Abad P (2010) Genome-wide survey and analysis of microsatellites in nematodes, with a focus on the plant-parasitic speciesmeloidogyne incognita. BMC Genom 11(1):598Google Scholar
  53. 53.
    Pasquesi GIM, Adams RH, Card DC et al (2018) Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals. Nat Commun 9(1):2774–2784Google Scholar
  54. 54.
    Castoe TA, Koning AP, Hall KT et al (2013) The Burmese python genome reveals the molecular basis for extreme adaptation in snakes. PNAS 110(51):20645–20650Google Scholar
  55. 55.
    Castoe TA, Hall KT, Mboulas MLG et al (2011) Discovery of highly divergent repeat landscapes in snake genomes using high-throughput sequencing. Genome Biol Evol 3(1):641–653Google Scholar
  56. 56.
    Sawaya S, Bagshaw A, Buschiazzo E, Kumar P, Chowdhury S, Black MA et al (2013) Microsatellite tandem repeats are abundant in human promoters and are associated with regulatory elements. PLOS ONE 8:e54710Google Scholar
  57. 57.
    Bhargava A, Fuentes FF (2010) Mutational dynamics of microsatellites. Mol Biotechnol 44(3):250–266Google Scholar
  58. 58.
    Schlötterer C (1998) Genome evolution: are microsatellites really simple sequences. Curr Biol 8(4):132–134Google Scholar
  59. 59.
    Ding S, Wang S, He K, Jiang M, Li F (2017) Large-scale analysis reveals that the genome features of simple sequence repeats are generally conserved at the family level in insects. BMC Genom 18(1):848Google Scholar
  60. 60.
    Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with nonrepetitive dna in plant genomes. Nat Genet 30(2):194–200Google Scholar
  61. 61.
    Fondon JW, Hammock EAD, Hannan AJ, King DG (2008) Simple sequence repeats: genetic modulators of brain function and behavior. Trends Neurosci 31(7):328–334Google Scholar
  62. 62.
    Francki M, Sharopova N (2008) Plant simple sequence repeats: distribution, variation, and effects on gene expression. Genome 51(2):79–90Google Scholar
  63. 63.
    Li YC, Korol A, Fahima T, Nevo E (2004) Microsatellites within genes: structure, function, and evolution. Mol Biol Evol 21(6):991–1007Google Scholar
  64. 64.
    Filiz E, Dogan I, Ozyigit II (2016) Analysis of EST-SSRS in silver birch (Betula pendula Roth.). J For Res 27(3):639–646Google Scholar
  65. 65.
    Yue BS, Li J, Hu TZ, Zhang XY, Li GZ, Jiang XM et al (2016) Distinct patterns of simple sequence repeats and GC distribution in intragenic and intergenic regions of primate genomes. Aging 8(11):2635–2650Google Scholar
  66. 66.
    Kelkar YD, Eckert KA, Chiaromonte F, Makova KD (2011) A matter of life or death: how microsatellites emerge in and vanish from the human genome. Genome Res 21(12):2038–2048Google Scholar
  67. 67.
    Metzgar D, Bytof J, Wills C (2000) Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Res 10(1):72–80Google Scholar
  68. 68.
    Lee HS, Park KU, Kim DW et al (2016) Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and microsatellite instability in patients with colorectal cancer and its clinical features. Curr Mol Med 16(9):829–839Google Scholar
  69. 69.
    Metzgar D, Wills C (2000) Evidence for the adaptive evolution of mutation rates. Cell 101(6):581–584Google Scholar
  70. 70.
    Metzgar D, Wills C, Trivedi S (2014) Analysis of simple sequence repeats in mammalian cell cycle genes. Recent Adv DNA Gene Seq 8(1):20–29Google Scholar
  71. 71.
    Chiang CM, Chien KY, Lin HJ et al (1996) Conformational change and inactivation of membrane phospholipid-related activity of cardiotoxin V from Taiwan cobra venom at acidic pH. Biochemistry 35(28):9167–9176Google Scholar
  72. 72.
    Gutierrez EM, Seebacher NA, Arzuman L et al (2016) Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT. Biochem Biophys Acta 7:1665–1681Google Scholar
  73. 73.
    Ulrich LE, Zhulin IB (2007) MiST: a microbial signal transduction database. Nucleic Acids Res 35:D386–D390Google Scholar
  74. 74.
    Zhu H, Guo L, Song P et al (2016) Development of genome-wide SSR markers in melon with their cross-species transferability analysis and utilization in genetic diversity study. Mol Breed 36(11):153–166Google Scholar
  75. 75.
    Kaldate R, Rana M, Sharma V et al (2017) Development of genome-wide SSR markers in horsegram and their use for genetic diversity and cross-transferability analysis. Mol Breeding 37:103–112Google Scholar
  76. 76.
    Poissant J, Shafer ABA, Davis CS et al (2009) Genome-wide cross-amplification of domestic sheep microsatellites in bighorn sheep and mountain goats. Mol Ecol Resour 9(4):1121–1126Google Scholar
  77. 77.
    Francisco LV, Langsten AA, Mellersh CS et al (1996) A class of highly polymorphic tetranucleotide repeats for canine genetic mapping. Mamm Genome 7(5):359Google Scholar
  78. 78.
    Sugita T, Semi Y, Sawada H, Utoyama Y, Hosomi Yuko (2013) Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum. Mol Breeding 31(4):909–920Google Scholar
  79. 79.
    Xu TJ, Sun DQ, Shi G, Wang RX (2010) Development and characterization of polymorphic microsatellite markers in the gray mullet (Mugil cephalus). Genet Mol Res 9(3):1791Google Scholar
  80. 80.
    Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32(3):314–331Google Scholar
  81. 81.
    Abdul-Muneer PM (2014) Application of microsatellite markers in conservation genetics and fisheries management: recent advances in population structure analysis and conservation strategies. Genet Res Int. Google Scholar
  82. 82.
    Garayalde AF, Poverene M, Cantamutto M, Carrera AD (2011) Wild sunflower diversity in Argentina revealed by ISSR and SSR markers: an approach for conservation and breeding programmes. Ann Appl Biol 158(3):305–317Google Scholar
  83. 83.
    Kumar M, Sharma VR, Kumar V, Sirohi U, Sharma S (2018) Genetic diversity and population structure analysis of indian garlic (Allium sativum L.) collection using SSR markers. Physiol Mol Biol Plants 25(2):377–386Google Scholar

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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life SciencesSichuan UniversityChengduChina
  2. 2.College of Materials and Chemistry & Chemical EngineeringChengdu University of TechnologyChengduChina
  3. 3.College of ForestryJiangxi Agricultural UniversityNanchangChina

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