شناسایی نواحی حاوی نشانگرهای EST-SSR در ترنسکریپتوم بلوط دارمازو (Quercus infectoria) با استفاده از فن‌آوری RNA-Seq

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشگاه لرستان

2 استاد، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران.

چکیده

هدف
دارمازو یا بلوط گال‌زا (Quercus infectoria) از درختان دارویی ارزشمند حاوی گال‌ها و تانن‌ها است. این گیاه یکی از گونه‌های جنس Quercus است که از زمان‌های قدیم به عنوان یک گیاه دارویی شناخته شده است. با وجود اهمیت فراوان این گونه، محدودیت اطلاعات ژنتیکی، مطالعات در مورد آن را دچار مشکل کرده است. نشانگرهای مولکولی EST-SSR، به دلیل داشتن تعداد بالای مکان‌های چندشکلی، می‌تواند اطلاعات ژنتیکی ویژه‌ای را ارائه دهد. در این مطالعه، نشانگرهای EST-SSR موجود در ترنسکریپتوم Q. infectoria که از سرهم بندی نوپدید خوانش های حاصل از RNA-Seq ایجاد شده، شناسایی شدند.
مواد و روش
از تکنیک توالی‌یابی RNA به منظور ایجاد سرهم‌بندی نوپدید استفاده و توالی‌یابی توسط پلتفرم Illumina HiSeq 2500 انجام شد. سرهم‌بندی نوپدید برای خوانش‌های با کیفیت بالا با استفاده از نرم‌افزار CLC Genomics Workbench (v7.5.0) انجام شد. به منظور تعیین نواحی نشانگرهای ریزماهواره ازMISA (ابزار جستجوی ریزماهواره) استفاده شد. در این مطالعه، ریزماهواره‌های تک نوکلئوتیدی تا شش نوکلئوتیدی شناسایی شدند. برای گروه‌بندی عملکردی ژن‌ها، آنالیز هستی شناسی ژن‌ها انجام شد. همچنین، نقشه‌برداری در برابر پایگاه‌های داده Plant Reactome به منظور شناسایی مسیرهای بیولوژیکی مرتبط با تک‌ژن‌های حاوی EST-SSR انجام شد.
نتایج
در این مطالعه، نواحی حاوی نشانگرهای EST-SSR در ترانسکریپتوم دارمازو که از سرهم‌بندی نوپدید خوانش‌های حاصل از RNA-Seq به دست آمده، شناسایی شدند. با بررسی 96225 یونی‌ژن از ترانسکریپتوم دارمازو، 20505 مورد ناحیه نشانگر EST-SSR در 13368 یونی‌ژن شناسایی شد که از این تعداد 4355 یونی‌ژن بیش از یک مکان نشانگر را دارا بودند. در بین نشانگرها، تکرار-های تک (مونو) نوکلئوتیدی (11394، 57/55 درصد) و پس از آن تکرار‌های دو و سه نوکلئوتیدی به‌ترتیب با 92/23 و 82/18 درصد دارای بیشترین فراوانی بودند. در مطالعه حاضر، بیشترین درصد موتیف‌های تک نوکلئوتیدی از نوع A/T، بیشترین موتیف‌های دی نوکلئوتیدی از نوع AG/CT (46/68 درصد) و بیشترین موتیف‌های 4،3، و 5 نوکلئوتیدی نیز به ترتیب از نوع AAG/CTT، AAAT/ATTT و AAAAG/CTTTT بودند. شناسایی مسیرهای فعال بیولوژیکی مرتبط با یونی‌ژن‌های دارای نشانگر با استفاده از پایگاه داده گیاهی انجام شد و بیشتر یونی‌ژن‌ها در مسیرهای انتقال سیگنال، متابولیسم پروتئین‌ها، چرخه سلولی، و بیان ژن (رونویسی) قرارگرفتند. بررسی مسیرهای غنی شده با یونی‌ژن‌های حاوی نشانگر نیز نشان داد که بیشترین فراوانی مربوط مسیرهای غشا، هسته و فرآیند متابولیکی ترکیبات حاوی نوکلئوبازها است.
نتیجه گیری: این مطالعه مجموعه‌ای از EST-SSR‌های مفید را ارائه می‌کند و می‌تواند زمینه تحقیقات عملکردی و مقایسه‌ای ژنومی بیشتر را بر روی دارمازو فراهم کند.

کلیدواژه‌ها

عنوان مقاله [English]

Identification of EST-SSR regions in the transcriptome of Quercus infectoria using RNA-Seq technology

نویسندگان [English]

  • Forough Joudaki 1
  • Ahmad Ismaili 2
  • Seyed Sajad Sohrabi 1
1 Faculty of Agriculture, Lorestan University, PO Box 465, Khor
2 Professor, Faculty of Agriculture, Lorestan University, Iran.
چکیده [English]

Objective
Gall oak (Quercus infectoria), is one of the extraordinary tree species and a valuable medicinal plant for its products, including galls and tannins. It is one of the most important species of the Quercus genus, which has been recognized and utilized as a medicinal plant since ancient times. Despite the importance of this species, limited basic genetic information has hindered research on this species. EST-SSR molecular markers, due to their high number of polymorphic loci, can provide specific genetic information. In this study, EST-SSR markers were identified in the Q. infectoria transcriptome generated through de novo assembly of RNA-Seq reads.
Materials and methods
The RNA sequencing technique was used for de novo transcriptome assembly and sequencing was performed by Illumina HiSeq 2500 platform. De-novo assembly was performed for high-quality reads using CLC Genomics Workbench (v7.5.0) software. For identification of microsatellite markers, MISA (Microsatellite searching Tool) was used. In this study, the microsatellites from mono-nucleotide to hexa-nucleotide were detected. To functionally group genes, GO analysis was performed. Also, mapping against the Plant Reactome pathway databases was performed to the identification of biological pathways associated with EST-SSR-containing unigenes.
Results
From 96,225 transcripts in the Q. infectoria transcriptome, 20505 EST-SSR markers were identified across 13368 unigenes, with 4,355 unigenes containing more than one marker locus. Among all types of repeats, mononucleotide repeats (11394, 55.57%) were the most abundant, followed by dinucleotide and three- nucleotide repeats with 23.92 and 18.82%, respectively. The highest percentage of mono-nucleotide motifs was A/T type, the highest percentage of di-nucleotide motifs was AG/CT type (68.46%), and the highest percentage of 3, 4, and 5 nucleotide motifs were AAG/CTT, AAAT/ATTT, and AAAAG/CTTTT type, respectively. Identification of biologically active pathways associated with SSR-containing unigenes was carried out using a plant database. Most unigenes were linked in signal transduction, protein metabolism, cell cycle, and gene expression (transcription) pathways. Analysis of the enriched pathways with SSR-containing unigenes also showed that the highest abundance was related to the membrane, nuclear, and metabolic pathways of compounds containing nucleobases.
Conclusions
This study provides valuable set of EST-SSRs for Q. infectoria and can provide the way for further functional and comparative genomic research on this species.

کلیدواژه‌ها [English]

  • EST-SSR marker
  • Quercus infectoria
  • RNA-Seq

مراجع

Ahmadi, A. J., & Ahmadikhah, A. (2022). Occurrence of simple sequence repeats in cDNA sequences of safflower (Carthamus tinctorius) reveals the importance of SSR-containing genes for cell biology and dynamic response to environmental cues. Frontiers in Plant Science, 13, Article 991107. https://doi.org/10.3389/fpls.2022.991107
Alaaddin Ahmed, A., Anwar Qadir, S., & Tahir, N. A.-R. (2022). CDDP and ISSR markers-assisted diversity and structure analysis in Iraqi Mazu (Quercus infectoria Oliv.) accessions. All Life, 15(1), 247–261. https://doi.org/10.1080/26895293.2022.2042401
Amilah, W. A. W. W. N., Mohamad, A. N., Izani, N. J. N., & Arizam, M. F. (2022). Antimicrobial activities of Quercus infectoria gall extracts: A scoping review. Journal of Herbal Medicine, 32, Article 100543. https://doi.org/10.1016/j.hermed.2022.100543
An, M., Deng, M., Zheng, S.-S., & Song, Y.-G. (2016). De novo transcriptome assembly and development of SSR markers of oaks Quercus austrocochinchinensis and Q. kerrii (Fagaceae). Tree Genetics & Genomes, 12, Article 1. https://doi.org/10.1007/s11295-016-1060-5
Askari, F., Azadi, A., Namavar-Jahromi, B., Tansaz, M., Mirzapour Nasiri, A., & Mohagheghzadeh, A. (2020). A comprehensive review about Quercus infectoria G. Olivier gall. Research Journal of Pharmacognosy, 7(1), 69–77. https://doi.org/10.22127/rjp.2019.184177.1494
da Fonseca, R. R., Albrechtsen, A., Themudo, G. E., Ramos-Madrigal, J., Sibbesen, J. A., Maretty, L., Zepeda-Mendoza, M. L., Campos, P. F., Heller, R., & Pereira, R. J. (2016). Next-generation biology: Sequencing and data analysis approaches for non-model organisms. Marine Genomics, 30, 3–13. https://doi.org/10.1016/j.margen.2016.04.012
Farahvashi, M., Mohammadabadi, M., Askari-Hesni, M., Amiri Ghanatsaman, Z., & Asadollahpour Nanaei, H. (2026). Genomic differentiation and diversity in Persian Gulf hawksbill turtles (Eretmochelys imbricata) revealed by the first whole-genome sequencing study. Animals, 16(2), Article 169. https://doi.org/10.3390/ani16020169
Filiz, E., & Kurt, F. (2021). Expression and co-expression analyses of WRKY, MYB, bHLH and bZIP transcription factor genes in potato (Solanum tuberosum) under abiotic stress conditions: RNA-seq data analysis. Potato Research, 64, 1–21. https://doi.org/10.1007/s11540-021-09502-3
Guerrero-Sanchez, V. M., Maldonado-Alconada, A. M., Amil-Ruiz, F., Verardi, A., Jorrín-Novo, J. V., & Rey, M.-D. (2019). Ion Torrent and Illumina, two complementary RNA-seq platforms for constructing the holm oak (Quercus ilex) transcriptome. PLOS ONE, 14(1), Article e0210356. https://doi.org/10.1371/journal.pone.0210356
Hajalizadeh, Z., Dayani, O., Khezri, A., Tahmasbi, R., Mohammadabadi, M., Solodka, T., Kalashnyk, O., Afanasenko, V., & Babenko, O. (2021). Expression of calpastatin gene in Kermani sheep using real-time PCR. Journal of Livestock Science and Technologies, 9(2), 51–57. https://doi.org/10.22103/jlst.2021.18165.1381
Hong, S.-Y., Roze, L. V., & Linz, J. E. (2013). Oxidative stress-related transcription factors in the regulation of secondary metabolism. Toxins, 5(4), 683–702. https://doi.org/10.3390/toxins5040683
Hosseini, S. Z., Ismaili, A., Nazarian Firouzabadi, F., Fallahi, H., & Rezaeinejad, A. (2018). Development of EST-SSR microsatellite markers related to drought tolerance in lentil (Lens culinaris). Crop Biotechnology, 8(23), 43–57.
Kalra, S., Puniya, B. L., Kulshreshtha, D., Kumar, S., Kaur, J., Ramachandran, S., & Singh, K. (2013). De novo transcriptome sequencing reveals important molecular networks and metabolic pathways of the plant, Chlorophytum borivilianum. PLOS ONE, 8(12), Article e83336. https://doi.org/10.1371/journal.pone.0083336
Kaur, G., Hamid, H., Ali, A., Alam, M. S., & Athar, M. (2004). Antiinflammatory evaluation of alcoholic extract of galls of Quercus infectoria. Journal of Ethnopharmacology, 90(2–3), 285–292. https://doi.org/10.1016/j.jep.2003.10.009
Khabiri, A., Toroghi, R., Mohammadabadi, M., & Tabatabaeizadeh, S. E. (2025). Whole genome sequencing and phylogenetic relative of a pure virulent Newcastle disease virus isolated from an outbreak in northeast Iran. Letters in Applied Microbiology, 78(4), Article ovaf049. https://doi.org/10.1093/lambio/ovaf049
Kim, K. K., Park, S. K., Kang, S. A., Park, M. K., Cho, M. K., Jung, H. J., Kim, K. K., & Yu, H. S. (2013). Comparison of functional gene annotation of Toxascaris leonina and Toxocara canis using CLC genomics workbench. The Korean Journal of Parasitology, 51(5), 525–530. https://doi.org/10.3347/kjp.2013.51.5.525
Li, Q., Su, X., Ma, H., Du, K., Yang, M., Chen, B., Fu, S., Fu, T., Xiang, C., & Zhao, Q. (2021). Development of genic SSR marker resources from RNA-seq data in Camellia japonica and their application in the genus Camellia. Scientific Reports, 11, Article 89350. https://doi.org/10.1038/s41598-021-89350-w
Li, Y. C., Korol, A. B., Fahima, T., Beiles, A., & Nevo, E. (2002). Microsatellites: Genomic distribution, putative functions and mutational mechanisms: A review. Molecular Ecology, 11(12), 2453–2465. https://doi.org/10.1046/j.1365-294X.2002.01643.x
Li, Y.-C., Korol, A. B., Fahima, T., & Nevo, E. (2004). Microsatellites within genes: Structure, function, and evolution. Molecular Biology and Evolution, 21(6), 991–1007. https://doi.org/10.1093/molbev/msh073
Liu, S., An, Y., Li, F., Li, S., Liu, L., Zhou, Q., Zhao, S., & Wei, C. (2018). Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (Camellia sinensis). Molecular Breeding, 38, Article 82. https://doi.org/10.1007/s11032-018-0824-z
López-Aljorna, A., Bueno, M. Á., Aguinagalde, I., & Martín, J. P. (2007). Fingerprinting and genetic variability in cork oak (Quercus suber L.) elite trees using ISSR and SSR markers. Annals of Forest Science, 64(7), 773–779. https://doi.org/10.1051/forest:2007057
Mohammadabadi, M. R., Nikbakhti, M., Mirzaee, H. R., Shandi, M. A., Saghi, D. A., Romanov, M. N., & Moiseyeva, I. G. (2010). Genetic variability in three native Iranian chicken populations of the Khorasan province based on microsatellite markers. Russian Journal of Genetics, 46(4), 505–509. https://doi.org/10.1134/S1022795410040198
Mohammadabadi, M., Afsharmanesh, M., Khezri, A., Kheyrodin, H., Babenko, O. I., Borshch, O., Kalashnyk, O., Nechyporenko, O., Afanasenko, V., Slynko, V., & Usenko, S. (2025). Effect of mealworm on GBP4L gene expression in the spleen tissue of Ross broiler chickens. Agricultural Biotechnology Journal, 17(2), 343–360. https://doi.org/10.22103/jab.2025.25277.1714
Palumbo, F., Vannozzi, A., Vitulo, N., Lucchin, M., & Barcaccia, G. (2018). The leaf transcriptome of fennel (Foeniculum vulgare Mill.) enables characterization of the t-anethole pathway and the discovery of microsatellites and single-nucleotide variants. Scientific Reports, 8(1), Article 10459. https://doi.org/10.1038/s41598-018-28775-2
Pereira-Leal, J. B., Abreu, I. A., Alabaça, C. S., Almeida, M. H., Almeida, P., Almeida, T., Amorim, M. I., Araújo, S., Azevedo, H., & Badia, A. (2014). A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing. BMC Genomics, 15(1), Article 371. https://doi.org/10.1186/1471-2164-15-371
Safari, M., Ismaili, A., Sohrabi, S. S., Nazarian-Firouzabadi, F., & Torabi Podeh, H. (2022). Transcriptome analysis of Persian oak (Quercus brantii L.) decline using RNA-seq technology. Biochemical Genetics, 60, 1–22. https://doi.org/10.1007/s10528-022-10283-8
Sajad Sohrabi, S., Ismaili, A., Azarian Firouz-Abadi, F. N., & Fallahi, H. (2018). Discovery of EST-SSRs markers in lentil (Lens culinaris) under cold stress. Crop Biotechnology, 8(22), 1–14.
Satya, P., Chakraborty, A., Jana, S., Majumdar, S., Karan, M., Sarkar, D., Datta, S., Mitra, J., Kar, C. S., & Karmakar, P. G. (2017). Identification of genic SSRs in jute (Corchorus capsularis, Malvaceae) and development of markers for phenylpropanoid biosynthesis genes and regulatory genes. Plant Breeding, 136(5), 784–797. https://doi.org/10.1111/pbr.12514
Sharopova, N. (2008). Plant simple sequence repeats: Distribution, variation, and effects on gene expression. Genome, 51(2), 79–90. https://doi.org/10.1139/G07-110
Shokri, S., Khezri, A., Mohammadabadi, M., & Kheyrodin, H. (2023). The expression of MYH7 gene in femur, humeral muscle and back muscle tissues of fattening lambs of the Kermani breed. Agricultural Biotechnology Journal, 15(2), 217–236. https://doi.org/10.22103/jab.2023.21524.1486
Shrestha, S., Kaushik, V. S., Eshwarappa, R. S. B., Subaramaihha, S. R., Ramanna, L. M., & Lakkappa, D. B. (2014). Pharmacognostic studies of insect gall of Quercus infectoria Olivier (Fagaceae). Asian Pacific Journal of Tropical Biomedicine, 4(1), 35–39. https://doi.org/10.1016/S2221-1691(14)60205-7
Soltani, N., Best, T., Grace, D., Nelms, C., Shumaker, K., Romero-Severson, J., Moses, D., Schuster, S., Staton, M., & Carlson, J. (2020). Transcriptome profiles of Quercus rubra responding to increased O₃ stress. BMC Genomics, 21(1), Article 6549. https://doi.org/10.1186/s12864-020-6549-5
Sun, J. J., Zhou, T., Zhang, R. T., Jia, Y., Zhao, Y. M., Yang, J., & Zhao, G. F. (2019). Comparative transcriptomes and development of expressed sequence tag-simple sequence repeat markers for two closely related oak species. Journal of Systematics and Evolution, 57(5), 440–450. https://doi.org/10.1111/jse.12469
Sun, L., Li, J., Sun, K., Wang, H., Yang, K., Chen, Q., & Lin, M. (2023). Development and characterization of EST-SSR markers in Rhodomyrtus tomentosa Hassk. based on transcriptome. Genetic Resources and Crop Evolution, 70(6), 1691–1705. https://doi.org/10.1007/s10722-022-01528-x
Taheri, S., Lee Abdullah, T., Yusop, M. R., Hanafi, M. M., Sahebi, M., Azizi, P., & Shamshiri, R. R. (2018). Mining and development of novel SSR markers using next generation sequencing (NGS) data in plants. Molecules, 23(2), Article 399. https://doi.org/10.3390/molecules23020399
Torre, S., Tattini, M., Brunetti, C., Fineschi, S., Fini, A., Ferrini, F., & Sebastiani, F. (2014). RNA-seq analysis of Quercus pubescens leaves: De novo transcriptome assembly, annotation and functional markers development. PLOS ONE, 9(11), Article e112487. https://doi.org/10.1371/journal.pone.0112487
Wang, D., Yang, T., Liu, R., Li, N., Wang, X., Sarker, A., Zhang, X., Li, R., Pu, Y., & Li, G. (2020). RNA-Seq analysis and development of SSR and KASP markers in lentil (Lens culinaris Medikus subsp. culinaris). The Crop Journal, 8(6), 953–965. https://doi.org/10.1016/j.cj.2020.04.007
Wang, L., Gong, X., Jin, L., Li, H., Lu, J., Duan, J., & Ma, L. (2019). Development and validation of EST-SSR markers of Magnolia wufengensis using de novo transcriptome sequencing. Trees, 33, 1213–1223. https://doi.org/10.1007/s00468-019-01853-2
Zhou, G.-L., & Zhu, P. (2020). De novo transcriptome sequencing of Rhododendron molle and identification of genes involved in the biosynthesis of secondary metabolites. BMC Plant Biology, 20(1), Article 2586. https://doi.org/10.1186/s12870-020-02586-y
Zhou, L., Sun, J., Zhang, T., Tang, Y., Liu, J., Gao, C., Zhai, Y., Guo, Y., Feng, L., & Zhang, X. (2022). Comparative transcriptome analyses of different Rheum officinale tissues reveal differentially expressed genes associated with anthraquinone, catechin, and gallic acid biosynthesis. Genes, 13(9), Article 1592. https://doi.org/10.3390/genes13091592
Zin, N. N. I. N. M., Rahimi, W. N. A. W. M., & Bakar, N. A. (2019). A review of Quercus infectoria (Olivier) galls as a resource for anti-parasitic agents: In vitro and in vivo studies. The Malaysian Journal of Medical Sciences, 26(6), 19–34. https://doi.org/10.21315/mjms2019.26.6.3
مجله بیوتکنولوژی کشاورزی
دوره 18، شماره 4
دی 1405
صفحه 23-44

فایل ها

هم رسانی

ارجاع به این مقاله

آمار