تمایز ژنوتیپ های نخود حساس و مقاوم به بیماری برق زدگی با استفاده از سامانه تکثیر انتخابی جهش

نوع مقاله : علمی پژوهشی-فارسی

نویسندگان

1 استادیار، گروه گیاه‌پزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

2 دانشیار، گروه گیاه‌پزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

10.22055/ppr.2024.45138.1714

چکیده

بیماری برق‌زدگی نخود یکی از عوامل مخرب و محدود‌کننده زراعت نخود در کشورهای مختلف می باشد. شناسایی منابع مقاومت با استفاده از روش‌های مولکولی مبتنی بر ردیابی ژنومی صفت مقاومت می‌تواند در تسریع و افزایش راندمان مؤثر باشد. استفاده از جهش نقطه‌ای کارکردی و آلل‌های مرتبط با مقاومت جهت ردیابی منابع مقاومت با استفاده از روش سامانه تکثیر انتخابی جهش (ARMS) می تواند در پیشبرد برنامه‌های گزینش منابع مقاومت به بیماری برق‌زدگی بسیار مؤثر باشد. قطعه دربرگیرنده جهش نقطه‌ای جایگاه GSh118-2773 با استفاده از آغازگرهای اختصاصی در دو ژنوتیپ نخود مقاوم (MCC133) و حساس (ILC263) به بیماری برق‌زدگی تکثیر و توالی‌یابی شد و حضور آلل‌های مرتبط به مقاومت GSh118-2773C و حساسیت GSh118-2773g به‌ترتیب در نمونه‌های مقاوم و حساس تأیید شد. جفت آغازگرهای اختصاصی ردیاب آلل مقاومتPSh18-Fc و PSh18-Rتوانست روی ژنوتیپ مقاوم MCC133 قطعه 330 جفت بازی مورد انتظار را تکثیر نماید. نتایج تکثیر اختصاصی آلل‌ها را با استفاده از آغازگرهای ردیابی تأیید نمود. استفاده از جفت آغازگرهای اختصاصی آلل مقاومت می تواند در مطالعات گزینش مولکولی منابع نخود مقاوم به بیماری برق‌زدگی مورد توجه قرار گیرد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Differentiation of susceptible and resistant chickpea genotypes to Ascochyta blight using the ARMS molecular method

نویسندگان [English]

  • F. Shokouhifar 1
  • M. Mamarabadi 2
1 Assistant Professor, Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
2 Associate Professor, Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
چکیده [English]

Abstract

Background and Objectives

Chickpea, cultivated in rainfed and irrigated fields across many countries, faces biological stresses such as Ascochyta blight caused by the pathogenic fungus Ascochyta rabiei. This disease poses a severe threat to chickpea fields, resulting in substantial annual damage in various countries. Identifying resistance sources through molecular methods, specifically genomic tracking of the resistance trait, holds the potential to expedite and enhance control measures effectively. The use of the Amplification Refractory Mutation System (ARMS) method, involving functional point mutations and resistance-related alleles, proves highly efficient in advancing selection programs targeted at combating Ascochyta blight.

Materials and methods

The fragment harboring the SNP18-Pos57723 point mutation within the GSh118 gene sequence underwent amplification and sequencing using PSh118-F/R specific primers. This process utilized DNA extracted from both resistant (MCC113) and susceptible (ILC263) chickpea genotypes. The identification of alleles associated with resistance and sensitivity was established. Distinct differentiating primers were formulated for resistant (PSh118-Fc and PSh118-R) and sensitive (PSh18-Fg and PSh18-R) genotypes. The specific annealing temperature for each reaction was determined through a temperature gradient analysis. The efficacy of the designed primers in distinguishing between resistant and sensitive genotypes was assessed by conducting PCR and comparing their electrophoresis patterns.

Results

The presence of alleles linked to GSh18-2773C resistance and GSh18-2773g sensitivity was confirmed in resistant and sensitive chickpea genotypes, respectively. The specific primers PSh18-Fc and PSh18-R, designed for detecting the resistance allele, successfully amplified the expected 330 bp fragment in the resistant chickpea genotype MCC133. The study results affirmed the accurate amplification of alleles using these designated primers.

Discussion

In the present study, the ARMS method, known for its high sensitivity, specificity, rapidity, cost-effectiveness, multiplexing capabilities, compatibility with high throughput methods, and non-destructive nature, was employed to differentiate chickpea genotypes resistant to Ascochyta blight. The ARMS method's effectiveness in selecting resistance sources among various plant lines and cultivars has been demonstrated through allele tracking related to the resistance gene in prior studies. The results of this study indicated that the ARMS method accurately distinguishes chickpea genotypes resistant and sensitive to Ascochyta blight based on alleles associated with the GSh18-2773 position's point mutation. Thus, employing specific primers for the resistance allele is recommended in molecular selection studies of chickpea sources resistant to Ascochyta blight.



Keywords: Point mutation, allelic variation, molecular markers, resistance, Ascochyta rabiei

کلیدواژه‌ها [English]

  • Point mutation
  • allelic variation
  • molecular markers
  • resistance
  • Ascochyta rabiei
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