Acta Oceanologica Sinica

, Volume 38, Issue 8, pp 41–47 | Cite as

Identification of SNP markers correlated with the tolerance of low-salinity challenge in swimming crab (Portunus trituberculatus)

  • Yanyan Feng
  • Dening Zhang
  • Jianjian Lv
  • Baoquan Gao
  • Jian LiEmail author
  • Ping Liu


Water salinity condition is an important factor for artificial propagation of the swimming crab (Portunus trituberculatus). Low salinity (LS)-resistant strains are preferred by crab industries. Single nucleotide polymorphism (SNP), the third generation of molecular markers, can be utilized in the breeding of LS-resistant species of P. trituberculatus. Our earlier study identified 615 genes differentially expressed in low-salinity stress compared to the controls. Although thousands of SNP loci have been found, it is hard to identify a SNP marker in correlation with a desired trait. In this study, time-of-flight mass spectrometry (TOF-MS), as an efficient method to select SNPs for the tolerance of LS challenge, was utilized for SNP typing. Fifty gene segments were amplified based on comparative transcriptomics in our earlier study, a total of 18 511 bp DNA fragments were amplified, and eighty-five SNP markers were found. The frequency of the SNPs was estimated to be 0.46 per 100 base pairs of DNA sequences. The rate of the conversion mutation was 81%, while the transversion mutation was 19%. The mutation rate of the G/T (C/A), A/T and G/C was 26%, 12% and 7%, respectively. Eight SNP markers were found to significantly correlate with the adaption of low salinity. Of the eight SNP markers, three linked-SNPs were found in the cuticle proportion gene, and another three SNPs were found in three new genes, and the rest two were found in aquaporin gene and chloride channel gene. The development of these SNP markers found in our study could be primarily used for breeding LS-resistant strains of P. trituberculatus.

Key words

Portunus trituberculatus low salinity time-of-flight mass spectrometry single nucleotide polymorphism SNP 


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We were grateful to all the laboratory members for experimental material preparation and technical assistance.


  1. Aitken N, Smith S, Schwarz C, et al. 2004. Single nucleotide polymorphism (SNP) discovery in mammals: a targeted-gene approach. Molecular Ecology, 13(6): 1423–1431, doi: 10.1111/j.1365-294x.2004.02159.xGoogle Scholar
  2. Arias A, Freire R, Boudry P, et al. 2009. Single nucleotide polymorphism for population studies in the scallops Aequipecten opercularis and Mimachlamys varia. Conservation Genetics, 10(5): 1491–1495, doi: 10.1007/s10592-008-9766-zGoogle Scholar
  3. Black IV W C, Baer C F, Antolin M F, et al. 2001. Population genomics: genome-wide sampling of insect populations. Annual Review of Entomology, 46: 441–469, doi: 10.1146/annurev.ento.46.1.441Google Scholar
  4. Bray W, Lawrence A L, Leung-Trujillo J. 1994. The effect of salinity on growth and survival of Penaeus vannamei, with observations on the interaction of IHHN virus and salinity. Aquaculture, 122(2-3): 133–146, doi: 10.1016/0044-8486(94)90505-3Google Scholar
  5. Chen R, Davydov E V, Sirota M, et al. 2010. Non-synonymous and synonymous coding SNPs show similar likelihood and effect size of human disease association. PLoS One, 5(10): e13574, doi: 10.1371/journal.pone.0013574Google Scholar
  6. Cui Zhaoxia, Liu Yuan, Wang Hongxia, et al. 2012. Isolation and characterization of microsatellites in Portunus trituberculatus. Conservation Genetics Resources, 4(2): 251–255, doi: 10.1007/s12686-011-9518-0Google Scholar
  7. Dai Aiyun, Feng Zhongqi, Song Yuzhi, et al. 1977. Primary investigation on the fishery biology of the Portunus trituberculatus. Chinese Journal of Zoology (in Chinese), (2): 30–33, doi: 10.13859/j.cjz.1977.02.015Google Scholar
  8. Dai Aiyun, Yang Siqiong, Song Yuzhi, et al. 1986. Marine Crabs in China Sea (in Chinese). Beijing: China Ocean Press, 194–195Google Scholar
  9. Germer S, Higuchi R. 1999. Single-tube genotyping without oligonucleotide probes. Genome Research, 9(1): 72–78Google Scholar
  10. Harding R M, Fullerton S M, Griffiths R C, et al. 1997. Archaic African and Asian lineages in the genetic ancestry of modern humans. American Journal of Human Genetics, 60(4): 772–789Google Scholar
  11. Hirschhorn J N, Sklar P, Lindblad-Toh K, et al. 2000. SBE-TAGS: an array-based method for efficient single-nucleotide polymorphism genotyping. Proceedings of the National Academy of Sciences of the United States of America, 97(22): 12164–12169, doi: 10.1073/pnas.210394597Google Scholar
  12. Holliday R, Grigg G W. 1993. DNA methylation and mutation. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 285(1): 61–67, doi: 10.1016/0027-5107(93)90052-HGoogle Scholar
  13. Ji Dongsheng. 2005. Techniques of pond-farming of swimming crab, Portunus trituberculatus. Special Economic Animal and Plant (in Chinese), 8(3): 12–13, doi: 10.3969/j.issn.1001-4713.2005.03.012Google Scholar
  14. Jin Yulin, Kong Lingfeng, Yu Hong, et al. 2014. Development, inheritance and evaluation of 55 novel single nucleotide polymorphism markers for parentage assignment in the Pacific oyster (Crassostrea gigas). Genes & Genomics, 36(2): 129–141, doi: 10.1007/s13258-013-0150-0Google Scholar
  15. Komar A A. 2007. SNPs, silent but not invisible. Science, 315(5811): 466–467, doi: 10.1126/science.1138239Google Scholar
  16. Kumlu M, Eroldogan O T, Saglamtimur B. 2001. The effects of salinity and added substrates on growth and survival of Metapenaeus monoceros (Decapoda: Penaeidae) post-larvae. Aquaculture, 196(1-2): 177–188, doi: 10.1016/S0044-8486(00)00580-9Google Scholar
  17. Kumlu M, Jones D A. 1995. Salinity tolerance of hatchery-reared postlarvae of Penaeus indicus H. Milne Edwards originating from India. Aquaculture, 130(2-3): 287–296, doi: 10.1016/0044-8486(94)00319-JGoogle Scholar
  18. Kwok P Y. 2001. Methods for genotyping single nucleotide polymorphisms. Annual Review of Genomics and Human Genetics, 2(2): 235–258, doi: 10.1146/annurev.genom.2.1.235Google Scholar
  19. Lai E, Riley J, Purvis I, et al. 1998. A 4-Mb high-density single nucleotide polymorphism-based map around human APOE. Genomics, 54(1): 31–38, doi: 10.1006/geno.1998.5581Google Scholar
  20. Li Xihong, Cui Zhaoxia, Liu Yuan, et al. 2013. Polymorphisms of antilipopolysaccharide factors in the swimming crab Portunus trituberculatus and their association with resistance/susceptibility to Vibrio alginolyticus. Fish & Shellfish Immunology, 34(6): 1560–1568, doi: 10.1016/j.fsi.2013.03.373Google Scholar
  21. Li Shuzhen, Wan Huirong, Ji Heyi, et al. 2009. SNP discovery based on CATS and genotyping in the finless porpoise (Neophocaena phocaenoides). Conservation Genetics, 10(6): 2013–2019, doi: 10.1007/s10592-009-9882-4Google Scholar
  22. Li W H, Sadler L A. 1991. Low nucleotide diversity in man. Genetics, 129(2): 513–523Google Scholar
  23. Livak K J, Marmaro J, Todd J A. 1995. Towards fully automated genome-wide polymorphism screening. Nature Genetics, 9(4): 341–342, doi: 10.1038/ng0495-341Google Scholar
  24. Lv Jianjian, Liu Ping, Wang Yu, et al. 2013. Transcriptome analysis of Portunus trituberculatus in response to salinity stress provides insights into the molecular basis of osmoregulation. PLoS One, 8(12): e82155, doi: 10.1371/journal.pone.0082155Google Scholar
  25. Ma Hongyu, Ma Qunqun, Ma Chunyan, et al. 2011. Isolation and characterization of gene-derived single nucleotide polymorphism (SNP) markers in Scylla paramamosain. Biochemical Systematics and Ecology, 39(4-6): 419–424, doi: 10.1016/j.bse.2011.05.024Google Scholar
  26. Morin P A, Aitken N C, Rubio-Cisneros N, et al. 2007. Characterization of 18 SNP markers for sperm whale (Physeter macrocephalus). Molecular Ecology Notes, 7(4): 626–630, doi: 10.1111/j.1471-8286.2006.01654.xGoogle Scholar
  27. Nickerson D A, Taylor S L, Weiss K M, et al. 1998. DNA sequence diversity in a 9. 7-kb region of the human lipoprotein lipase gene. Nature Genetics, 19(3): 233–240, doi: 10.1038/907Google Scholar
  28. Péqueux A. 1995. Osmotic regulation in crustaceans. Journal of Crustacean Biology, 15(1): 1–60, doi: 10.1163/193724095X00578Google Scholar
  29. Petrov D A, Hartl D L. 1999. Patterns of nucleotide substitution in Drosophila and mammalian genomes. Proceedings of the National Academy of Sciences of the United States of America, 96(4): 1475–1479, doi: 10.1073/pnas.96.4.1475Google Scholar
  30. Piatek A S, Tyagi S, Pol A C, et al. 1998. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nature Biotechnology, 16(4): 359–363, doi: 10.1038/nbt0498-359Google Scholar
  31. Rafalski A. 2002. Applications of single nucleotide polymorphisms in crop genetics. Current Opinion in Plant Biology, 5(2): 94–100, doi: 10.1016/S1369-5266(02)00240-6Google Scholar
  32. Rouse D B, Kartamulia I. 1992. Influence of salinity and temperature on molting and survival of the Australian freshwater crayfish (Cherax tenuimanus). Aquaculture, 105(1): 47–52, doi: 10.1016/0044-8486(92)90160-mGoogle Scholar
  33. Ruscoe I M, Shelley C C, Williams G R. 2004. The combined effects of temperature and salinity on growth and survival of juvenile mud crabs (Scylla serrata Forskål). Aquaculture, 238(1-4): 239–247, doi: 10.1016/j.aquaculture.2004.05.030Google Scholar
  34. Sauvage C, Bierne N, Lapègue S, et al. 2007. Single Nucleotide polymorphisms and their relationship to codon usage bias in the Pacific oyster Crassostrea gigas. Gene, 406(1-2): 13–22, doi: 10.1016/j.gene.2007.05.011Google Scholar
  35. Schütz E, Von Ahsen N, Oellerich M. 2000. Genotyping of eight thiopurine methyltransferase mutations: three-color multiplexing, “two-color/shared” anchor, and fluorescence-quenching hybridization probe assays based on thermodynamic nearestneighbor probe design. Clinical Chemistry, 46(11): 1728–1737Google Scholar
  36. Shen L X, Basilion J P, Stanton V P Jr. 1999. Single-nucleotide polymorphisms can cause different structural folds of mRNA. Proceedings of the National Academy of Sciences of the United States of America, 96(14): 7871–7876, doi: 10.1073/pnas.96.14.7871Google Scholar
  37. Smith C T, Elfstrom C M, Seeb L W, et al. 2005. Use of sequence data from rainbow trout and Atlantic salmon for SNP detection in Pacific salmon. Molecular Ecology, 14(13): 4193–4203, doi: 10.1111/j.1365-294X.2005.02731.xGoogle Scholar
  38. Sommer S S, Groszbach A, Bottema C. 1992. PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single-base changes. Biotechniques, 12(1): 82–87Google Scholar
  39. Soyel H I, Kumlu M. 2003. The effects of salinity on postlarval growth and survival of Penaeus semisulcatus (Decapoda: Penaeidae). Turkish Journal of Zoology, 27(3): 221–225Google Scholar
  40. Stickney H L, Schmutz J, Woods I G, et al. 2002. Rapid mapping of zebrafish mutations with SNPs and oligonucleotide microarrays. Genome Research, 12(12): 1929–1934, doi: 10.1101/gr.777302Google Scholar
  41. Storey J D, Tibshirani R. 2003. Statistical significance for genomewide studies. Proceedings of the National Academy of Sciences of the United States of America, 100(16): 9440–9445, doi: 10.1073/pnas.1530509100Google Scholar
  42. Syvänen A C. 1999. From gels to chips: “minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms. Human Mutation, 13(1): 1–10, doi: 10.1002/(SICI)1098-1004(1999)13:1<1:AID-HUMU1>3.0.CO;2-IGoogle Scholar
  43. Taillon-Miller P, Gu Zhijie, Li Qun, et al. 1998. Overlapping genomic sequences: a treasure trove of single-nucleotide polymorphisms. Genome Research, 8(7): 748–754, doi: 10.1101/gr.8.7.748Google Scholar
  44. Tran H T T, Takeshima Y, Surono A, et al. 2005. A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro. Molecular Genetics and Metabolism, 85(3): 213–219, doi: 10.1016/j.ymgme.2005.03.006Google Scholar
  45. Wang Jun, Chuang Karen, Ahluwalia M, et al. 2005. High-throughput SNP genotyping by single-tube PCR with Tm-shift primers. Biotechniques, 39(6): 885–893, doi: 10.2144/000112028Google Scholar
  46. Wang D G, Fan Jianbing, Siao C J, et al. 1998. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science, 280(5366): 1077–1082, doi: 10.1126/science.280.5366.1077Google Scholar
  47. Xue Junzeng, Du Nanshan, Lai Wei, et al. 1997. A review of studies on Portunus trituberculatus in China. Donghai Marine Science (in Chinese), 15(4): 60–65Google Scholar
  48. Yu Yang, Wei Jiankai, Zhang Xiaojun, et al. 2014. SNP discovery in the transcriptome of White Pacific Shrimp Litopenaeus vannamei by next generation sequencing. PLoS One, 9(1): e87218, doi: 10.1371/journal.pone.0087218Google Scholar

Copyright information

© Chinese Society for Oceanography and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yanyan Feng
    • 1
  • Dening Zhang
    • 1
  • Jianjian Lv
    • 1
  • Baoquan Gao
    • 1
  • Jian Li
    • 1
    Email author
  • Ping Liu
    • 1
  1. 1.Key Laboratory of Sustainable Development of Marine Fisheries of Ministry of Agriculture, Yellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdaoChina

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