In collaboration with Payame Noor University and Iranian Biotechnology Society

Document Type : Research Paper

Authors

Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran

Abstract

Thyme species are very important due to the production of secondary metabolites such as terpenoids. Since the identification of key genes such as genes related to terpenoids biosyntesis pathway can play an effective role in plant breeding programs, especially thyme species, the present study was aimed to investigate the transcriptomes of T. daenensis, T. vulgaris, T. lancifolius, T. persicus, T. pubescens to identify key genes in the biosynthesis of monoterpenoids, chloroplast genes sequence and evaluation of similarities and differences among these species. For this purpose, total RNAs extracted from vegetative growth were sent to Macrogene of Korea for sequencing with theIllumina HiSeq 2500 platform. After assembling the sequences using various tools, the best results was selected and transcripts were documented in different databases. Then, according to the documented results, key genes responsible in the synthesis of terpenoids and chloroplast gene sequence were identified, and then phylogenetic relationships among species was investigated. According to the evaluation indicators, the best assembly was a product of Binpacker tools. Based on the results, the sequence of 10 genes involved in the synthesis of terpenoids was obtained. Interestingly, among the identified TPSs, most of the contigs were classified into the TPSb and TPSa classes of terpenoids. The sequence of 73 chloroplast genes was extracted from the transcriptome data and finally the phylogenetic relationship was evaluated according to 400, 70 bp of cpDNA. The study of phylogenetic relationships showed a close genetic relationship between T. daenensi and T.vulgaris which can introduce T. daenensis as an appropriate replacement for T. vulgaris in different purpose, especially in pharmacological applications. The results show that Z. multiflora can most probably be as one of the ancestors of Thymus, which is significantly different from Thymus species in terms of its genetic structure, especially the key genes of the terpene biosynthesis pathway.

Keywords

Main Subjects

Abdolahinia, E. D., Bashir, N. S., Hagnazari, A., Nazemiyeh, H., & Hejazi, M. S. (2011). A comparative phenotypic and ITS based genotypic study in thyme species (Thymus L. Lamiaceae). Vegetos, 24(2), 102-113. Andrews, S., Krueger, F., Seconds-Pichon, A., Biggins, F., & Wingett, S. (2015). FastQC. A quality control tool for high throughput sequence data. Babraham Bioinformatics. Babraham Inst. Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114-2120. Bräuchler, C., Meimberg, H., & Heubl, G. (2010). Molecular phylogeny of Menthinae (Lamiaceae, Nepetoideae, Mentheae)–taxonomy, biogeography and conflicts. Molecular phylogenetics and evolution, 55(2), 501-523. Bryant, D. M., Johnson, K., DiTommaso, T., Tickle, T., Couger, M. B., Payzin-Dogru, D., ... & Whited, J. L. (2017). A tissue-mapped axolotl de novo transcriptome enables identification of limb regeneration factors. Cell reports, 18(3), 762-776. Bunsawat, J., Elliott, N.E., Hertweck, K.L., Sproles, E. and Alice, L.A. (2004). Phylogenetics of Mentha (Lamiaceae): evidence from chloroplast DNA sequences. Systematic botany, 29(4), 959-964. Capella-Gutiérrez, S., Silla-Martínez, J. M., & Gabaldón, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 25(15), 1972-1973. Drew, B. T., & Sytsma, K. J. (2012). Phylogenetics, biogeography, and staminal evolution in the tribe Mentheae (Lamiaceae). American journal of botany, 99(5), 933-953. Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic acids research, 32(5), 1792-1797. Federici, S., Galimberti, A., Bartolucci, F., Bruni, I., De Mattia, F., Cortis, P. & Labra, M. (2013). DNA barcoding to analyse taxonomically complex groups in plants: the case of Thymus (Lamiaceae). Botanical Journal of the Linnean Society, 171(4), 687-699. Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., & Regev, A. (2011). Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature biotechnology, 29(7), 644-652. Ghobadi, S., Marouf, A. & Majd, M. (2016). Differential expression of the key genes involved in the biosynthesis of monoterpenes in different tissues and in response to abiotic elicitors in Summer savory (Satureja hortensis). Cell and Tissue Journal, 7, 3. Habibi, S., Ehteshamnia, A., Fatehi, F. & Ghaderi, A. (2020). Investigation of γ-Terpinene synthesis and α- Terpinene synthesis reductoisomerase genes expression and its relation to monoterpene carvacrol biosynthesis in Thymus vulgaris cv. ‘Varico 3’. Genetic Engineering and Biosafety Journal, 9(2), 161-169 Haji Akhondi, A. & Farahani Kia, B. (1386). What do you know about thyme?. Encyclopedia of Medicinal Plants, 12: 12-9. (in persian) Izadiyan, P., Hemmateenejad, B., Mohsen Taghavi, S., & Izadiyan, M. (2018). Discrimination of Shirazi thyme from thymus species and antioxidant activity prediction using chemometrics and FT-IR spectroscopy. Journal of the Iranian Chemical Society, 15, 259-268. Jamzad, Z. (1373). Thyme. Tehran: Research Institute of Forests and Pastures of the country. (in persian) Jamzad, Z. (1388). Iranian herbs and spices. Tehran: Research Institute of Forests and Pastures of the country. (in persian) Jiang, S. Y., Jin, J., Sarojam, R., & Ramachandran, S. (2019). A comprehensive survey on the terpene synthase gene family provides new insight into its evolutionary patterns. Genome biology and evolution, 11(8), 2078-2098. Kumar, S., Stecher, G., Suleski, M., & Hedges, S. B. (2017). TimeTree: a resource for timelines, timetrees, and divergence times. Molecular biology and evolution, 34(7), 1812-1819. Liu, J., Li, G., Chang, Z., Yu, T., Liu, B., McMullen, R., ... & Huang, X. (2016). BinPacker: packing-based de novo transcriptome assembly from RNA-seq data. PLoS computational biology, 12(2), e1004772. Mashhady Malekzadeh, A Majdi, M. & Maroufi, A. (2017). Expression analysis of biosynthetic genes of thymol and carvacrol in different tissues of thyme (Thymus vulgaris). Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 25(1), 160-171. Mojiri, F., Esmaeili, A., Nazarian Firouz Abadi, F., Maddahi Arefi, H., & Ahmadi, H. (2014). Study of genetic diversity of Iranian Thymus accessions, using ISJ semi-random markers. Agricultural Biotechnology Journal, 6(2), 147-162. (in persian) Omidbeigi, R. (1393). Production and processing of medicinal plants. Mashhad: Astan Quds Razavi. (in persian) Priya, P., Yadav, A., Chand, J., & Yadav, G. (2018). Terzyme: a tool for identification and analysis of the plant terpenome. Plant Methods, 14, 1-18. Quinlan, A. R., & Hall, I. M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6), 841-842. Rambaut, A. & Drummond, A. J. (2014). BEAST 2: A software platform for Bayesian evolutionary analysis. PLoS Comput. Biol, 10, e1003537 Rambaut, A., & Drummond, A. J. (2018). FigTree v1.4.0 in Computer Program and Documentation Distributed by the Author. 2012. Available online: http://tree.bio.ed.ac.uk/software/figtree/. Rana, S. B., Zadlock IV, F. J., Zhang, Z., Murphy, W. R., & Bentivegna, C. S. (2016). Comparison of de novo transcriptome assemblers and k-mer strategies using the killifish, Fundulus heteroclitus. PloS one, 11(4), e0153104. Rustaiee, A. R., Yavari, A., Nazeri, V., Shokrpour, M., Sefidkon, F., & Rasouli, M. (2013). Genetic diversity and chemical polymorphism of some Thymus species. Chemistry & Biodiversity, 10(6), 1088-1098. Rustaiee, A., Sefidkon, F., Tabatabaei, S. M. F., Omidbaigi, R., & Mirahmadi, S. F. (2011). Chemical polymorphism of essential oils from five populations of Thymus daenensis Celak. subsp. daenensis endemic to Iran. Journal of Essential Oil Research, 23(3), 6-11. Salzberg, S. L., Phillippy, A. M., Zimin, A., Puiu, D., Magoc, T., Koren, S., & Yorke, J. A. (2012). GAGE: A critical evaluation of genome assemblies and assembly algorithms. Genome research, 22(3), 557-567. Schliesky, S., Gowik, U., Weber, A. P., & Bräutigam, A. (2012). RNA-seq assembly–are we there yet?. Frontiers in plant science, 3, 220. Smith-Unna, R., Boursnell, C., Patro, R., Hibberd, J. M., & Kelly, S. (2016). TransRate: reference-free quality assessment of de novo transcriptome assemblies. Genome research, 26(8), 1134-1144. Smolik, M., Jadczak, D. and Korzeniewska, S. )2009(. Assessment of morphological and genetic variability in some Thymus accessions using molecular markers. Not. Bot. Hortic. Agrobo. 37: 234-240. Sonboli, A., Mirjalili, M. H., Bakhtiar, Z. I. B. A., & Jamzad, Z. I. B. A. (2013). Molecular authentication of Thymus persicus based on nrDNA ITS sequences data. Iran J. Bot, 19, 179-185. Thompson, J. D., Manicacci, D., & Tarayre, M. (1998). Thirty-five years of thyme: a tale of two polymorphisms. BioScience, 48(10), 805-815.