PCR-SSCP: a method to fingerprinting sequence variability in plant gene pool using the Iranian wild diploid Triticum gene pool as a case study

Document Type : Scientific Reports

Authors

• Fatemeh Nasernakhaei
• Mohammad Reza Rahiminejad
• Hojjatollah Saeidi
• Manoochehr Tavassoli

University of Isfahan, Departement of Biology

https://doi.org/10.22067/jcmr.v6i1.31099

Abstract

As a case study, the genotypic polymorphism of the Iranian material of diploid Triticum was screened based on internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA) using single strand conformation polymorphism (SSCP) analysis. This is a simple and cost effective technique for genotyping and investigating the allelic polymorphism among plant populations. Performing SSCP analysis among 21 accessions, two distinctive migration profiles associated with the two validly recognized diploid Triticum species i.e., T. monococcum subsp. aegilopoides and T. urartu were detected. Nucleotide sequencing confirmed the distinction of two types of observed profiles. This study suggested that SSCP is an applicable diagnostic molecular tool for screening the genotypic variability before the costly direct genomic sequencing is begun.

Keywords

• diploid
• Iran
• ITS2
• SSCP
• Triticum

20.1001.1.20089147.2014.6.1.4.8

References

1- Blatter R. H. E., Jacomet S. and Schlumbaum A. (2002) Spelt-specific alleles in HMW glutenin genes from modern and historical European spelt (Triticum spelta L.) Theoretical and Applied Genetics 104, 2–3: 329–337.

2- Bodenes C., Laigret F. and Kremer A. (1996) Inheritance and molecular variations of PCR-SSCP fragment in Pedunculate oak (Quercus robur L.). Theoretical and Applied Genetics 93, 3: 348–354.

3- Borchert T. and Hohe A. (2009) Identification of molecular markers for the flower type in the ornamental crop Calluna vulgaris. Euphytica 170, 1–2: 203–213.

4- Castelblanco W. and Fregene M. (2006) SSCP-SNP-based conserved ortholog set (COS) markers for comparative genomics in cassava (Manihot esculenta crantz). Plant Molecular Biology Reporter 24, 2: 229–236.

5- Chen S., Yao H., Han J., Liu C., Song J., Shi L., Zhu, Y., Ma X., Gao T., Pang X., Luo K., Li Y., Li X., Jia X., Lin Y. and Leon C. (2010) Validation of the ITS2 region as a novel DNA Barcode for identifying medicinal plant species. Public Library of Science ONE 5: e8613.

6- Cotton R. G. H. (1997) Slowly but surely towards better scanning for mutations. Trends in Genetics 13, 2: 43–46.

7- Crautlein M., Korpelainen H., Pietilainen M. and Rikkinen J. (2011) DNA barcoding: a tool for improved taxon identification and detection of species diversity. Biodiversity and Conservation 20, 2: 373389.

8- Dumolin-Lapegue S., Bodenes C. and Petit R. J. (1996) Detection of rare polymorphisms in mitochondrial and of oaks with PCR-RFLP combined to SSCP analysis. Forest Genetics 3, 4: 227230.

9- Ebihara A., Ishikawa H., Matsumoto S., Lin S. J., Iwatsuki K., Takamiya N., Watano Y. and Ito M. (2005) Nuclear DNA, chloroplast DNA, and ploidy analysis clarified biological complexity of the Vandesbochia radicans complex (Hymenophyllaceae) in Japan and adjacent areas. American Journal of Botany 92, 9: 15351547.

10- Fukuoka S., Inoue T., Miyao A., Monna L., Zhong H. S., Sasaki T. and Minobe, Y. (1994) Mapping of sequence-tagged sites in rice by single strand conformation polymorphism. DNA Research 1, 6: 271277.

11- Gasser R. B. and Chilton N. B. (2001) Applications of single-strand conformation polymorphism (SSCP) to taxonomy, diagnosis, population genetics and molecular evolution of parasitic nematodes. Veterinary Parasitology 101, 34: 201213.

12- Gasser R. B., Hu M., Chilton N. B., Campbell B. E., Jex A. J., Otranto D., Cafarchia C., Beveridge I. and Zhu X. (2006) Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation. Nature Protocols 1, 6: 31213128.

13- Gawel N. J. and Jarret R. L. (1991) A modified CTAB extraction procedure for Musa and Ipomoea. Plant Molecular Biology Reporter 9, 3: 262266.

14- Hall T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series 41: 95–98.

15- Hayashi K. (1991) PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR methods and applications 1, 1: 3438.

16- Karatas D. D., Kunter B., Coppola G. and Velasco R. (2010) Analysis of polymorphism based on SSCP markers in gamma-irradiated (Co60) grape (Vitis vinifera) varieties. Genetics and Molecular Research 9, 4: 23572363.

17- Kita Y., and Ito M. (2000) Nuclear ribosomal ITS sequences and phylogeny in East Asian Aconitum subgenus Aconitum (Ranunculaceae), with special reference to extensive polymorphism in individual plants. Plant Systematics and Evolution 225, 14: 113.

18- Kuhn D. N., Motamayor J. C., Meerow A. W. and Schnell R. J. (2008) SSCP markers provide a useful alternative to microsatellites in genotyping and estimating genetic diversity in populations and germplasm collections of plant specialty crops. Electrophoresis 29, 19: 40964108.

19- Lu Y., Curtiss J., Percy R. G., Hughs S. E., Yu S. and Zhang J. (2009) DNA polymorphisms of genes involved in fiber development in a selected set of cultivated tetraploid cotton. Crop Science 49, 5: 16951704.

20- Martins-Lopes P., Zhang H. and Koebner R. (2001) Detection of single nucleotide mutations in wheat using single strand conformation polymorphism Gels. Plant Molecular Biology Reporter 19, 2: 159162.

21- Meusnier I., Valero M., Destombe C., Gode C., Desmarais E., Bonhomme F., Stam W. T. and Olsen J. (2002) PCR-SSCP analyses of nuclear and chloroplast DNA provide evidence for recombination, multiple introductions and nascent speciation in the Caulerpa taxifolia complex. Molecular Ecology 11: 23172325.

22- Olivares-Fuster O., Hernandez-Garrido M., Guerri J. and Navarro L. (2007) Plant somatic hybrid cytoplasmic DNA characterization by single-strand conformation polymorphism. Tree Physiology 27, 6: 785792.

23- Orita M., Suzuki Y., Sekiya T. and Hayashi K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 4: 874879.

24- Plomion C., Hurme P., Frigerio J. M., Ridolfi M., Pot D. and Pionneau C. (1999) Developing SSCP markers in two Pinus species. Molecular Breeding 5, 1: 2131.

25- Rahiminejad M. R. and Kharazian N. (2005) Illustration of and a key to the genus Triticum L. in Iran. Wheat information Service 99: 2934.

26- Rodriguez F., Cai D., Teng Y. and Spooner D. (2011) Asymmetric single-strand conformation polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants. American Journal of Botany 98, 7: 1061–1067.

27- Rousseau-Gueutin M., Gaston A., Ainouche A., Ainouche M. L., Olbricht K., Staudt G., Richard L. and Denoyes-Rothan B. (2009) Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics Evolution 51, 3: 515530.

28- Salimi A., Ebrahimzadeh H. and Taeb M. (2005) Description of Iranian diploid wheat resources. Genetic Resources and Crop Evolution 52, 4: 351361.

29- Salmaso M., Faes G., Segala C., Stefanini M., Salakhutdinov I., Zyprian E., Toepfer R., Grando M. S. and Velasco R. (2004) Genome diversity and gene haplotypes in the grapevine (Vitis vinifera L.), as revealed by single nucleotide polymorphisms. Molecular Breeding 14, 4: 385395.

30- Sanguinetti C. J., Dias Neto E. and Simpson A. J. G. (1994) Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17, 5: 915–919.

31- Sato Y. and Nishio T. (2003) Mutation detection in rice waxy mutants by PCR-RF-SSCP. Theoretical and Applied Genetics 107, 3: 560567.

32- Sunnucks P., Wilson A. C. C., Beheregaray L. B., Zenger K., French J. and Taylor A. C. (2000) SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Molecular Ecology 9, 11: 16991710.

33- Liberado P. and Rozas J. (2009) DnaSP version 5.10: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451–1452.

34- Swapna M., Sivaraju K., Sharma R. K., Singh N. K. and Mohapatra T. (2011) Single strand conformational polymorphism of EST-SSRs: A potential tool for diversity analysis and varietal identification in sugarcane. Plant Molecular Biology Reporter 29, 3: 505513.

35- To K. Y., Liu C. I., Liu S. T. and Chang Y. S. (1993) Detection of point mutations in the chloroplast genome by single-strand conformation polymorphism analysis. The Plant Journal 3, 1: 183186.

36- Wang G. Z., Miyashita N. T. and Tsunewaki K. (1997) Plasmon analyses of Triticum (wheat) and Aegilops: PCR-single-stranded conformational polymorphism (PCR-SSCP) analyses of organellar DNAs. Proceedings of the National Academy of Science of USA 94, 26: 1457014577.

37- Watano Y., Imazu M. and Shimizu T. (1995) Chloroplast DNA typing by PCR-SSCP in the Pinus pumila, P. parviflora var. pentaphylla complex (Pinaceae). Journal of Plant Research 108, 4: 493499.

38- Watano Y., Kanai A. and Tani N. (2004) Genetic structure of hybrid zones between Pinus pumila and P. parviflora var. pentaphylla (Pinaceae) revealed by molecular hybrid index analysis. American Journal of Botany 91, 1: 6572.

39- White T. J., Bruns T. D., Lee S. B. and Taylor J. W. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR-protocols and applications-a laboratory manual. (Eds. Innis, N., Gelfand, D., Sninsky, J. and White, J.) Academic Press, New York, 315322 pp.

40- Yao H., Song J., Liu C., Luo K., Han J., Li Y., Pang P., Xu H., Zhu Y., Xiao P. and Chen S. (2010) Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals. Public Library of Science ONE 5: e13102.