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Evaluation of five genomic DNA extraction methods for downstream molecular applications in fresh and herbarium leaves of Astragalus fridae | ||
| Rostaniha | ||
| دوره 24، شماره 2 - شماره پیاپی 69، اسفند 2023، صفحه 89-98 اصل مقاله (367.66 K) | ||
| نوع مقاله: Research Paper | ||
| شناسه دیجیتال (DOI): 10.22092/bot.j.iran.2023.360969.1343 | ||
| نویسندگان | ||
| Zahra Alidoosty Shahraky1؛ Atefe Amirahmadi* 2؛ Parisa Farrokh3؛ Arezou Rezaei3؛ Javad Ghasemian4 | ||
| 1MSc Graduate in Biochemistry, School of Biology, Damghan University, Damghan, Iran | ||
| 2Assistant Prof., School of Biology and Institute of Biological Sciences, Damghan University, P.O. Box 36716-45667, Damghan, Iran | ||
| 3Assistant Prof., School of Biology and Institute of Biological Sciences, Damghan University, Damghan, Iran | ||
| 4Assistant Prof., School of Mathematics and Computer Sciences, Damghan University, Damghan, Iran | ||
| چکیده | ||
| The protection of genetic resources is crucial for safeguarding national assets as natural resources face various threats such as climate change and changing land use. To conserve endangered species such as Astragalus fridae, an endemic species of gypsum soils of Semnan province (Iran) which is on the brink of extinction due to limited distribution and habitat destruction, establishing a gene bank is essential. To ensure the survival of this species, it is necessary to extract high-quality and high-quantity DNA. The present survey, has evaluated five genome extraction methods including Protocol A (main CTAB), Protocol B (CTAB without β-mercaptoethanol), Protocol C (CTAB without ammonium acetate), Protocol D (the modified Murray & Thompson method), and Protocol E (GeneAll Plant Kit), to determine their ability to extract DNA from fresh and herbarium leaves of A. fridae. The quality and quantity of DNA were assessed using gel electrophoresis, spectrophotometry, and usability of the purified DNA tested by PCR of the ITS region of nuclear ribosomal DNA. Protocol D yielded the highest quality of the extracted genome from fresh leaves, and Protocol A, extracted the highest DNA concentration from fresh leaves. Although Protocols B and C extracted a reasonable amount of genome of acceptable quality with dry leaves, the ITS region was not amplified in these samples, rendering them unsuitable for DNA extraction of A. fridae. | ||
| کلیدواژه‌ها | ||
| climate change؛ CTAB؛ endangered species؛ endemic؛ threats | ||
| عنوان مقاله [English] | ||
| ارزیابی پنج روش استخراج DNA ژنومی برای کاربردهای مولکولی پایین دست در برگ‌های تازه و هرباریومی Astragalus fridae | ||
| نویسندگان [English] | ||
| زهرا علیدوستی شهرکی1؛ عاطفه امیراحمدی2؛ پریسا فرخ3؛ آرزو رضایی3؛ جواد قاسمیان4 | ||
| 1دانش‌آموخته کارشناسی ارشد بیوشیمی، دانشکده زیست شناسی، دانشگاه دامغان، دامغان، ایران | ||
| 2استادیار دانشکده زیست شناسی و پژوهشکده علوم زیستی، دانشگاه دامغان، کدپستی 45667-36716، دامغان، ایران | ||
| 3استادیار دانشکده زیست شناسی و پژوهشکده علوم زیستی، دانشگاه دامغان، دامغان، ایران | ||
| 4استادیار دانشکده ریاضی و علوم کامپیوتر، دانشگاه دامغان، دامغان، ایران | ||
| چکیده [English] | ||
| حفاظت از ذخایر ژنتیکی جزو حیاتی حفاظت از سرمایه ملی است. از آنجایی که منابع طبیعی توسط عوامل مختلفی از جمله تغییرات آب و هوایی و تغییر کاربری اراضی تهدید می‌شود، بنابراین ایجاد بانک ژن برای حفاظت از گونه‌های در خطر انقراض ضروری به نظر می‌رسد. Astragalus fridae Rech.f. (باقلاییان) گونه بومی خاک‌های گچی استان سمنان است که به دلیل پراکنش محدود و تخریب زیستگاه در حال انقراض می‌باشد. برای اطمینان از بقای این گونه، باید تلاش‌هایی برای استخراج DNA با کیفیت و کمیت بالا انجام شود. به این منظور، پنج روش استخراج ژنوم شامل پروتکل A (CTAB اصلی)، پروتکل B (CTAB بدون بتا-مرکاپتواتانول)، پروتکل C (CTAB بدون استات آمونیوم)، پروتکل D (روش اصلاح شده موری و تامسون) و پروتکل E (کیت گیاهی GeneAll)، از نظر توانایی آن‌ها در استخراج DNA از برگ‌های تازه و هرباریومی مورد ارزیابی قرار گرفتند. کیفیت و کمیت DNA توسط ژل الکتروفورز و اسپکتروفتومتری بررسی شد و قابلیت استفاده DNA خالص شده توسط PCR ناحیه ITS DNA ریبوزومی هسته‌ای مورد آزمایش قرار گرفت. بالاترین کیفیت ژنوم استخراج شده به پروتکل D با برگ‌های تازه اختصاص یافت. بیشترین غلظت DNA از برگ‌های تازه (پروتکل A) به دست آمد. اگرچه مقدار معقولی از ژنوم با کیفیت قابل قبول توسط پروتکل‌های B و C با برگ‌های خشک استخراج شد ولی منطقه ITS در این نمونه‌ها تکثیر نشد، بنابراین پروتکل‌های B و C برای استخراج ژنوم A. fridae مناسب نبودند. | ||
| کلیدواژه‌ها [English] | ||
| بومی, تغییرات آب و هوایی, تهدید, در معرض خطر انقراض, CTAB | ||
| مراجع | ||
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