شناسایی نشانگرهای EST-SSR عدس (Lens culinaris) تحت تنش سرما

نوع مقاله : علمی پژوهشی

نویسندگان

1 دانشجوی دکتری گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

2 دانشیار گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

3 استاد گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

4 استادیار گروه زیست شناسی، دانشکده علوم پایه، دانشگاه رازی، کرمانشاه، ایران

چکیده

توسعه برنامه‌های به‌نژادی عدس برای مقابله با عوامل نامساعد محیطی نسبت به سایر حبوبات، به علت کمبود منابع ژنتیکی با چالش‌های بیشتری روبرو می‌باشد. نشانگرهای EST-SSR به‌دلیل ایجاد چندشکلی در نواحی کدکننده ژن‌ها، یکی از پرکاربردترین نشانگرهای مولکولی در بسیاری از برنامه‌های اصلاحی به‌شمار می‌روند. از این‌رو در تحقیق حاضر به‌منظور توسعه نشانگرهای EST-SSR در مقیاس بالا، از فرآیند سرهم‌بندی مجموعه خوانش‌های کوتاه حاصل از فناوری توالی‌یابی RNA عدس تحت تنش سرما و حالت طبیعی استفاده شد. به‌منظور اعمال تنش سرما، گیاهچه‌های 21 روزه عدس به‌مدت 48 ساعت در معرض دمای 4 درجه سانتی‌گراد قرار گرفتند. از نمونه‌های گیاهی تهیه شده، RNA کل استخراج شد و مورد توالی‌یابی قرار گرفتند. تعداد 8905 مکان ریزماهواره در 7211 یونی‎ژن حاصل از داده‌های RNA-seq عدس شناسایی شد که 1293 یونی‌ژن شامل بیش از یک مکان نشانگر SSR بود. فراوان‌ترین نوع نشانگر EST-SSR یافت شده از نوع تک نوکلئوتیدی بود. در این مطالعه موتیف‌های A/T، AG/CT و AAG/CTT به‌ترتیب بیشترین فراوانی را در بین موتیف‌های تک، دو و سه نوکلئوتیدی به خود اختصاص دادند. نتایج بلاست یونی‌ژن‌های حاوی SSR، نشان داد که 80 درصد از یونی‌ژن‌ها دارای رکورد مشابه در پایگاه پروتئین‌های غیرتکراری بودند. تفسیر کارکردی ژن‌ها، حضور یونی‌ژن‌های حاوی ریزماهواره را در زیر‌گروه‌های مهم پاسخ به تنش سرما نظیر اتصال، سلول و اجزای سلول و متابولیک را نشان داد. همچنین با توجه به نتایج می‌توان چنین اظهار نمود که اغلب نشانگرهای شناسایی شده در ژن‌هایی قرار داشتند که نقش مهمی در پاسخ به تنش‌ سرما دارند و جایگاه احتمالی اغلب آن‌ها نواحی UTR می‌باشد. از این‌رو بررسی بیشتر این نواحی در رونوشت‌ ژن‌های کاندید پاسخ‌دهنده به تنش سرما از اهمیت بیشتری برخوردار می‌باشد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Discovery of EST-SSRs Markers in Lentil (Lens culinaris) under Cold Stress

نویسندگان [English]

  • Seyed Sajad Sohrabi 1
  • Ahmad Ismaili 2
  • Farhad N azarian Firouz-Abadi 3
  • Hossein Fallahi 4
1 Ph.D. Student, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
2 Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
3 Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
4 Assistant Professor, Department of Biology, Faculty of Basic Sciences, Razi University, Kermanshah, Iran
چکیده [English]

Development and improvement of lentil breeding programs to deal with adverse environmental factors in comparison to other legumes has more challenges due to poor pool of genetic resources. EST-SSR (EST-Simple Sequence Repeats) markers are one of the most commonly used molecular markers in many plant breeding programs due to polymorphism in genes coding regions. Hence, in the present study, for the development of EST-SSR markers, assembly process of RNA sequences of lentil under cold stress and normal condition was used. In order to apply cold stress, lentil plants treated at 4 °C condition. Total RNA was extracted from plant samples and was sequenced. 8905 microsatellite locations in 7211 unigene derived from lentil RNA sequences data was identified that 1293 unigene of them contained more than one SSR marker location. The most abundant type of EST-SSR marker was found to be of single nucleotide type. In this study, A/T, AG/CT and AAG/CTT motifs had the highest frequency among the one, two and three nucleotide motifs, respectively. The results of blast of unigene containing SSR showed that 80% of the unigenes had a similar record in the non-redundant protein database. The functional annotation of the unigenes showed that unigene containing SSR marker are subordinate to the critical stress-responsive terms such as binding, cell, cell parts and metabolic. Also, according to the results of this study, it can be stated that most of the identified EST-SSR markers were found in genes that play an important role in responding to cold stress, and UTR regions is often possible position. Hence, more analysis of these areas in candidate gene transcripts in response to cold stress is more important.

کلیدواژه‌ها [English]

  • Cold stress
  • Lentil
  • Tri-nucleotide repeat motifs
  • EST-SSR markers

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