Journal of Vegetables Sciences

Journal of Vegetables Sciences

Identification of Ecotypes and Different Species of Savory (Satureja spp.) Using DNA Barcoding Genes

Document Type : Original Article

Authors
Tahereh Peiri 1
arash fazeli 2
1 M. Sc. Graduted, Agriculture and Plant Breeding Ilam University, Ilam, Iran
2 Assistant Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Ilam University, Ilam, Iran
10.22034/iuvs.2022.540180.1179
Abstract
Extended Abstract

Introduction: Medicinal plants are valuable resources that have been considered by developing and developed countries considered as raw materials to become safe drugs for humans. Satureja belonging to the Lamiaceae This genus has more than 91 species with a wide distribution in the Mediterranean region. There are more than 8 species of this genus in Iran. Genetic identification and registration of different plant cultivars is one of the important stages of protecting genetic resources, which will be a difficult task for most plants in the early stages due to morphological characteristics. DNA barcoding is an emerging technology for fast and accurate species identification, and it is a molecular-based identification system that has recently been widely used, and its purpose is to identify biological samples and assign them to a specific species. Molecular DNA barcoding method is a reliable tool for identifying medicinal plants at the genus and species level. In this technique, a small part of the plant genome is sequenced and used to identify and determine the relationship between different species of a genus.
Materials and Methods: In the present study, the seeds of different species of savory plants including ( horentiss, S. bakhtiari, S. biossier, S. mutica, S. bakhtiari) were prepared from different regions of Ilam province and the National Center of Genetic and Biological Resources of Iran were investigated using DNA barcoding. Seed cultivation was done in April 2016 in the greenhouse of Ilam University. The seeds of the used species were grown in small pots. After the seeds germinated, when the seedlings grew to the stage of two to three leaves, the young leaves were sampled for DNA extraction. Genomic DNA was extracted by Doyle et al.'s method in the biotechnology laboratory of Ilam University in 1397. Quality and quantity of genomic DNA was checked using agarose gel 1/2% and nanodrop device respectively. PCR was performed using specific primers for matK, rbcL and ITS genes. Among the studied genes only matK gene was amplified in all samples.  After purification of PCR products, their product were sequenced. In order analysis of data, first, the sequence Blast in NCBI database using nucleotide Blast. Alignment of sequence was done using Clustalw method by BioEdite Sequence Alignment Editor Software. Indeed, Phylogenetic tree created using MEGA-X software.
Results and Discussion: PCR amplification show that the only matK gene well amplified in all samples, while the other genes were not amplified.  Also, the quality of the graphs shows the high quality of the sequence of the amplified fragment of the PCR product and the purity of the examined samples.  Nucleotide BLAST in NCBI show that our results have high similarity with related spices in NCBI (more than 96%). The results of the analysis of content of nucleotides in the studied samples in comparison with the sample in NCBI show that according to the changes observed in the length of the fragment amplified on the agarose gel, but there were no significant changes in the molecular percentage of nucleotides in the studied samples. Cluster analysis based on UPGMA method of matK gene showed that the samples of biossier and S. bakhtiarica of Ilam and Gilan along with the sample of S. hortensis Birjand and Yazd were in the same group and samples of s. bakhtiari Yazd and s. Khorasan mutica were in the second group.
Conclusion: The results of PCR amplification show that matK gene has been amplified in all samples that it can be very well way for investigating and determining of phylogenetic relationship of different savory species. The results of matK gene sequence as one of the candidate genes in plant DNA barcodes showed that the studied samples and ecotypes along with samples extracted from NCBI were classified into three groups. The results of this study showed that it is possible to use the matK gene as a part of the barcode system to identify savory species. However, due to the lack of information about the barcode library for the matK gene in the Satureja genus, it is not possible to make a definite conclusion about the species. The matK gene has two advantages, including the efficient yield of quality sequences and a high level of species discrimination. In general, re-sequencing of matK gene can be separated different samples from each other based on geographical origin. 
Keywords
Satureja spp
Medicinal plant
DNA barcoding
Sequencing

Asadi, F., Dezhsetan, S., Ghahremanzadeh, R., Razmjo, J. & Alebrahim, M T. (2015). DNA barcoding of some local medicinal plants of Ardabil province. Crop Biotechnology. 5(10), 31-40. (In persian).

Asahina, H., Shinozaki, J., Masuda, K., Morimitsu, Y. and Satake, M. (2010). Identification of medicinal Dendrobium species by phylogenetic analyses using matK and rbcL sequences.  Journal of Natural Medicines, 64, 133-138.
Bafeel, S. O., Arif, I. A., Bakir, M. A., Khan, H. A., Al Farhan, A. H., Al Homaidan, A. A., ... & Thomas, J. (2011). Comparative evaluation of PCR success with universal primers of maturase K (matK) and ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (rbcL) for barcoding of some arid plants. Plant Omics Journal, 4(4), 195-198.
Burgess, K.S., Fazekas, A.J., Kesanakurti, P.R., et al (2011). Discriminating plant species in a local temperate flora using the rbcL+ matK DNA barcode. Methods in Ecology and Evolution, 2, 333-340.
Caceres, M.E., Pupilli, F., Piano, E., Arcioni, S. (2000). RFLP markers are an effective tool for the identification of creeping bentgrass (Agrostis stolonifera L.) cultivars. Genetic Resources and Crop Evolution, 47, 455-459.
Casiraghi, M., Labra, M., Ferri, E., Galimberti, A., De Mattia, F. (2010).DNA barcoding: a six-question tour to improve users’ awareness about the method. Briefings in Bioinformatics, 11, 440- 453.
Chen, S., Yao, H., Han, J., Liu, C., Song, J. (2010). Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS one, 5 (1), e8613.
Dong W, Liu J, Yu J, Wang L, et al. (2012). Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding. PLoS One, 7, e35071.
Doyle, J.J. & Doyle J.L. (1990).  Isolation of plant DNA from fresh tissue. Focus, 12, 13-15.

Esmaeili, H., Hadian, J., Mirjalili, M H. & Rezadost, H. (2016). Evaluation of Some Morphological, Phytochemical and Ecological Characteristics of Different Populations of Satureja rechingeri Jamzad. Journal of Range and Watershed Management, 69(1), 1-12. (In Farsi).

Felsenstein, J. (2004). Inferring phylogenies, Publishers Sunderland Massachusetts, 1-645.
Gao, T., Yao, H., Song, J., Liu, C., Zhu, Y., Ma, X., & Chen, S. (2010). Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2. Journal of ethnopharmacology, 130(1), 116-121.
Gulluce, M., Sokmen, M., Daferera, D., Agar, G., Ozkan, H., Kartal, N., Polissiou, M., Sokmen, A. and Sahin, F. (2003). In vitro antibacterial, antifungal, and extracts of herbal parts and callus cultures of Satureja hortensis L. Journal of Agricultural and Food Chemistry, 51 (14), 3958-3965.
Hatari, Z., Zamani, Z., Nazeri, V. & Tabrizi, L. (2013). Evaluation of genetic diversity in the medicinal plant Kakooti ( Ziziphora tenuior L.) using morphological characteristics and RAPD markers. Modern Genetic Journal, 8(1). 19-28. (In Farsi).
Hebert, P. D., Cywinska, A., Ball, S. L., & DeWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B. Biological Sciences, 270(1512), 313-321.
Hebert, P. D., Stoeckle, M. Y., Zemlak, T. S., & Francis, C. M. (2004). Identification of birds through DNA barcodes. PLoS biology, 2(10), e312.
Jing, Y.U., Jian-Hua, X. U. E., and Shi-Liang, Z. H. O. U. (2011). New universal matK primers for DNA barcoding angiosperms. Journal of Systematics and Evolution. 49 (3), 176–181.
Kress, W.J., Erickson, D.L., (2007). A two-locus global DNA barcode for land plants: The coding rbcL gene complements the noncoding trnH-psbA spacer region. PloS one. 2, e508.
 
Kuo LY, Li FW, Chiou WL, Wang CN (2011) First insights into fern matK phylogeny. Molecular  Phylogenetics and  Evolution.  59, 556-566.
Manen, JF., Habashi, C., Jeanmonod, D., Park, JM, Schneeweiss, GM. (2004) Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences. Molecular Phylogenetics and Evolution, 33, 482-500.
Mohr, G., Perlman, P. S., and Lambowitz, A. M. (1993). Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function. Nucleic Acid Research, 21, 4991-4997.
Mount, D.M. (2004). Bioinformatics: sequence and Genome Analysis 2nd ed. Cold Spring Harbor Laboratory Press: NY.
Müller KF, Borsch T and Hilu KW (2006). Phylogenetic utility of rapidly evolving DNA at high taxonomical levels: contrasting matK, trnT-F, and rbcL in basal angiosperms. Molecular  Phylogenetics and  Evolution. 41, 99-117.
Omidbaigi, R., Production and Processing of Medicinal Plant. (2000). Austan Quds publication. (In Farsi).
Pang, X., Shi, L., Song, J., Chen, X., & Chen, S. (2013). Use of the potential DNA barcode ITS2 to identify herbal materials. Journal of natural medicines, 67(3), 571-575.
Sanders, E. R., Karol, K. G., and McCourt, R. M. (2003). Occurrence of matK in a trnK group II intron in charophyte green algae and phylogeny of the Characeae. American  Journal of  Botany. 90(4), 628-633.
Schori, M., and Showalter, A. M. (2011). DNA barcoding as a means for identifying medicinal plants of Pakistan. Pakistan Journal of Botany, 43, 1-4.
Sefidkon, F., Jamzad, Z., and Barazandeh, M.M. (2005). Essential oil of Satureja bachtiarica Bunge. A potential source of carvacrol. Iranian Journal of Medicinal and Aromatic Plants, 20 (4), 425-439.
Tamura K., &  Nei M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10,512-526.
Techen, N., Parveen, I., Pan, Z., & Khan, I. A. (2014). DNA barcoding of medicinal plant material for identification. Current Opinion in Biotechnology, 25, 103-110.
Theodoridis S, Stefanaki A, Tezcan M, Aki C, Kokkini S, Vlachonasios KE. 2012. DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çes¸me-Karaburun Peninsula (Turkey). Molecular Ecology Resources 12, 620–633.
Vanisree, M., Lee, C. Y., Lo, S. F., Nalawade, S. M., Lin, C. Y., and Tsay, H. S. (2004). Studies on the production of some important secondar metabolites from medicinal plants by plant tissue cultures. Botanical Bulletin- Academia Sinica Taipei, 45(1),1- 22.
Zuo, Y., Chen, Z., Kondo, K., Funamoto, T., Wen, J. and Zhou, S. (2011) DNA Bar-coding of Panax Species. Planta Medica, 77, 182-187.

  • Receive Date 02 October 2021
  • Revise Date 06 January 2022
  • Accept Date 29 January 2022