| تعداد نشریات | 285 |
| تعداد نشریات سازمان | 83 |
| تعداد شمارهها | 3,133 |
| تعداد مقالات | 34,859 |
| تعداد مشاهده مقاله | 50,316,670 |
| تعداد دریافت فایل اصل مقاله | 90,223,778 |
Evaluation of different method of DNA extraction from smut fungi teliospores | ||
| Mycologia Iranica | ||
| دوره 12، شماره 2، اسفند 2025، صفحه 123-131 اصل مقاله (1.06 M) | ||
| نوع مقاله: Short Article | ||
| شناسه دیجیتال (DOI): 10.22092/mi.2025.370368.1322 | ||
| نویسندگان | ||
| Hossein Kargar؛ Bahram Sharifnabi* | ||
| Department of Plant Protection, College of Agriculture, Isfahan University of Technology, Isfahan, IRAN | ||
| چکیده | ||
| Smut fungi, particularly those within the genera Tilletia, Ustilago, Sporisorium, and Urocystis, threaten cereal crops worldwide. Accurate molecular identification of these pathogens requires high-quality genomic DNA, however, extracting DNA from smut teliospores remains a challenge due to their thick and resilient cell walls. To address this, four commonly used DNA extraction methods — Murray & Thompson, Raeder & Broda, Chelex 100 and HotSHOT — were comparatively evaluated across ten species of smut fungi: Tilletia laevis, T. caries, T. controversa, T. indica, Ustilago tritici, U. nuda, U. hordei, Sporisorium maydis, S. ehrenbergii, and Urocystis agropyri. Mechanical disruption using sterilized carborundum, with or without liquid nitrogen, was applied uniformly across all methods. The quality and quantity of extracted DNA were assessed by Nanodrop spectrophotometry and PCR amplification using ITS1/ITS4 primers. All methods yielded PCR-amplifiable DNA; however, substantial differences were observed in yield and purity. Raeder & Broda method yielded the highest average DNA concentration (1050.55 ng/μl), followed by HotSHOT (951.33 ng/μl), Murray & Thompson (918.82 ng/μl), and Chelex (879.37 ng/μl). While HotSHOT offered a higher yield than Murray & Thompson and Chelex, its lower purity suggested co-purification of cellular contaminants. Murray & Thompson and Raeder and Broda-extracted DNA exhibited better overall purity based on their average A260/A280 and A260/A230 ratios. Electrophoresis of PCR products revealed strong and consistent bands for all samples, indicating successful DNA amplification. This study demonstrates that Murray & Thompson remains optimal when purity is crucial, while Raeder & Broda is effective for higher-yield applications. HotSHOT and Chelex, despite lower purity, remain useful for rapid and routine diagnostics. | ||
| کلیدواژهها | ||
| Bunts and smuts؛ Chelex؛ HotSHOT؛ Thick wall spore | ||
| مراجع | ||
|
Durán R, Fischer GW. 1961. The Genus Tilletia. Washington State University Press, Pullman, WA, USA.
Fernandez JA, Durán R. 1978. Hypodermic inoculation, a rapid technique for producing dwarf bunt in wheat. Plant Disease Reporter. 62: 336-337.
Fuentes-Dávila G, Goates BJ, Thomas P, Nielsen J, Ballantyne B. 2002. Smut diseases. In: Bread Wheat: Improvement and Production (Curtis BC, Rajaram S, Gómez Macpherson H. eds). Food and Agriculture Organization of the United Nations, Vialle delle Terme di Caracalla, Rome, Italy. https://www.fao.org/4/y4011e/y4011e0h.htm#bm17
Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes‐application to the identification of mycorrhizae and rusts. Molecular ecology. 2: 113-118.
Goates BJ. 1996. Common bunt and dwarf bunt. In: Bunt and Smut Diseases of Wheat: Concepts and Methods of Disease Management (Hettel GP, McNab A. eds): 12-25. CIMMYT. Mexico.
Grey WE, Mathre DE, Hoffmann JA, Powelson RL, Fernandez JA. 1986. Importance of seedborne Tilletia controversa for infection of winter wheat and its relationship to international commerce. Plant Disease. 70: 122-125.
Lindner DL, Banik MT. 2011. Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus. Mycologia. 103:731-740.
Murray MG, Thompson WF. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research. 8(19): 4321-4321.
Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson KH. 2008. Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evolutionary bioinformatics. 4(4): 193-201.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, … et al. 2000. Loop-mediated isothermal amplification of DNA. Nucleic acids research. 28(12): e63.
Raeder U, Broda P. 1985. Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology. 1(1): 17-20.
Simon UK, Weiß M. 2008. Intragenomic variation of fungal ribosomal genes is higher than previously thought. Molecular biology and evolution. 25: 2251-2254.
Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, … et al. 2000. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and Tris (HotSHOT). BioTechniques. 29(1): 52-54.
Walsh PS, Metzger DA, Higuchi R. 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques. 10(4): 506-513.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications. (Innis MA, Gelfand JJ, Sninsky DH and White TJ, eds): 315-322. Academic Press, USA.
| ||
|
آمار تعداد مشاهده مقاله: 160 تعداد دریافت فایل اصل مقاله: 152 |
||