Evaluation of five genomic DNA extraction methods for downstream molecular applications in fresh and herbarium leaves of Astragalus fridae

Document Type : Research Paper

Authors

1 MSc Graduate in Biochemistry, School of Biology, Damghan University, Damghan, Iran

2 Assistant Prof., School of Biology and Institute of Biological Sciences, Damghan University, P.O. Box 36716-45667, Damghan, Iran

3 Assistant Prof., School of Biology and Institute of Biological Sciences, Damghan University, Damghan, Iran

4 Assistant Prof., School of Mathematics and Computer Sciences, Damghan University, Damghan, Iran

10.22092/bot.j.iran.2023.360969.1343

Abstract

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.

Keywords

Article Title [Persian]

ارزیابی پنج روش استخراج DNA ژنومی برای کاربردهای مولکولی پایین دست در برگ‌های تازه و هرباریومی Astragalus fridae

Authors [Persian]

  • زهرا علیدوستی شهرکی 1
  • عاطفه امیراحمدی 2
  • پریسا فرخ 3
  • آرزو رضایی 3
  • جواد قاسمیان 4
1 دانش‌آموخته کارشناسی ارشد بیوشیمی، دانشکده زیست شناسی، دانشگاه دامغان، دامغان، ایران
2 استادیار دانشکده زیست شناسی و پژوهشکده علوم زیستی، دانشگاه دامغان، کدپستی 45667-36716، دامغان، ایران
3 استادیار دانشکده زیست شناسی و پژوهشکده علوم زیستی، دانشگاه دامغان، دامغان، ایران
4 استادیار دانشکده ریاضی و علوم کامپیوتر، دانشگاه دامغان، دامغان، ایران
Abstract [Persian]

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.

Keywords [Persian]

  • بومی
  • تغییرات آب و هوایی
  • تهدید
  • در معرض خطر انقراض
  • CTAB

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History

  • Receive Date: 29 March 2023
  • Revise Date: 17 May 2023
  • Accept Date: 31 May 2023

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