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شناسایی و بررسی تنوع ژنتیکی ویروس لکه‌حلقوی بافت مرده هسته‌دارها (Prunus necrotic ring spot virus) در درختان میوه هسته‌دار و دانه‌دار استان کردستان

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی سابق کارشناسی ارشد، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه کردستان، سنندج، ایران

2 استادیار، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه کردستان، سنندج، ایران

3 استادیار پژوهش، موسسه تحقیقات ثبت و گواهی بذر و نهال، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران

چکیده

شمار 149 نمونۀ برگی از درختان میوۀ هسته‌دار و دانه‌دار استان کردستان هنگام بهار و تابستان سال‌های 1394 و 1396 به‌منظور شناسایی مولکولی و بررسی گوناگونی ژنتیکی عامل لکه حلقوی بافت مردۀ درختان هسته‌دار جمع‌آوری شدند و با آغازگرهای اختصاصی ژن پروتئین پوششی PNRSV-F3/PNRSV-R3 آزمون RT-PCR شدند. نتیجه‏ها RT-PCR نشان دادند که 8/20 درصد از نمونه‌ها آلوده به PNRSV بودند. سیزده جدایه بر پایۀ نوع میزبان و منطقۀ جغرافیایی گزینش و پس از تکثیر و پیوست بخشی از ژن پروتئین پوششی آن‌ها به پلاسمید pTG-19 و همسانه‌سازی در باکتری E. coli تعیین توالی شدند. توالی‌های به‌دست‌آمده در سطح نوکلئوتیدی به‌طور میانگین 002/0 ± 9/98 درصد با یکدیگر و 006/0 ± 4/94 درصد با دیگر جدایه‌های موجود در بانک ژن همانندی نوکلئوتیدی نشان دادند. در واکاوی تبارزایی بر پایۀ ترادف نوکلئوتیدی جدایه‌های PNRSV در سه گروه تبارزایی PV96، PV32 و PE5 قرار گرفتند که سیزده جدایۀ این پژوهش به همراه بیشتر جدایه‌های ایرانی در گروه تبارزایی PV96 قرار گرفتند. بیشترین همانندی نوکلئوتیدی میان جدایه‌های هلو از کامیاران (KH10)، زردآلو از سنندج (SZ93) و هلو از دهگلان (D7) با جدایۀ شلیل از ایران (KX353935) با 98 درصد و کمترین همانندی نوکلئوتیدی میان جدایۀ زردآلو از سنندج (SZ26) با جدایۀ آلو از لهستان (DQ983499) با 6/83 درصد همانندی دیده شد. نسبت‌های کم جانشینی مترادف به غیر مترادف (dN/dS) در ژن پروتئین پوششی این ویروس روشنگر این نکته است که گزینش منفی نقش بزرگی را در فرگشت این ژن بازی کرده است و بررسی نوترکیبی با نرم‌افزار RDP v.4.63 نیز نشان داد که در جدایه‌های موردبررسی در این پژوهش نوترکیبی در این بخش از ژنوم رخ نداده است.

کلیدواژه‌ها


عنوان مقاله [English]

Phylogenetic analysis of Prunus necrotic ring spot virus in pome and stone fruit trees in Kurdistan province

نویسندگان [English]

  • Mahdi Azaryar 1
  • Mohammad Hajizadeh 2
  • Abdolbaset Azizi 2
  • masoud Naderpour 3
1 Former M.Sc. Student, Department of Plant Pathology, University of Kurdistan, Sanandaj, Iran
2 Assistant Professor, Department of Plant Pathology, University of Kurdistan, Sanandaj, Iran
3 Assistant Professor, Seed and Plant Improvement Institute, Education and Extension Organization, Karaj, Iran
چکیده [English]

In order to study molecular identification and genetic diversity of Prunus necrotic ringspot virus (PNRSV), 149 leaf samples from 37 orchards of pome and stone fruit trees of Kurdistan province were collected and tested by RT-PCR using a PNRSV-F3/PNRS-R3 specific primer pair. Results showed that 20.8% of the samples were infected by PNRSV.In the next stage of the study, 13 isolateswere selected based on the host and geographic region and their amplified fragments were ligated into the pTG19-T plasmid, transformed to E. coli DH5α, and sequenced. Phylogenetic analysis showed that PNRSV isolates formed three clades PV96, PV32, and PE5, and all new-sequences from Kurdistan (in this research) were categorized in PV96 phylogenetic clade, which is close to other Iranian isolates. The new isolates shared 99 ± 0.002 identities together and 94/9 ± 0.005 with other previously PNRSV reported isolates at the nucleotide level. Pairwise comparisons of sequences showed that isolates peach from Kamyaran (KH10), pear from Sanandaj (SZ93), and peach from Dehgolan (D7) had the highest nucleotide similarity (98%) with Iranian isolate nectarine (KX353935) whereas isolate pear from Sanandaj (SZ26) had the lowest nucleotide identity with isolate plum from Poland (DQ983499). The low dN/dS ratio in all populations of the virus showed that negative selection plays important role in PNRSV-CP evolution and recombination. There is no recombination event in this domain of PNRSV genome of these isolates.

کلیدواژه‌ها [English]

  • Coat protein
  • negative selection
  • recombination
  • Phylogenetic analysis
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