Investigation of Viral Agents in Walnut (Juglans spp.) Trees by High Throughput Sequencing from Niğde province, Türkiye

Bahar Makbule Temeltürk; Serkan Önder; Çiğdem Ulubaş Serçe

doi:10.18016/ksutarimdoga.vi.1618751

EN TR

Investigation of Viral Agents in Walnut (Juglans spp.) Trees by High Throughput Sequencing from Niğde province, Türkiye

Abstract

In October 2023, virus-like symptomatic walnut trees in Niğde province were investigated using high-throughput sequencing (HTS) to investigate viral infections. Double-stranded RNA (dsRNA) extraction was conducted, followed by conversion of dsRNA into cDNA. PCR was then performed using barcoding with MID primers for metagenomic analyses. The PCR products from walnut samples B13, B22, B23, and B21 + B5 were sequenced using the Illumina NovaSeq 6000 sequencing platform. The sequencing data underwent bioinformatics analysis using Geneious Prime and CLC Genomic Workbench. In this comparison, more contigs were constructed in CLC Genomic Workbench, resulting in more precise outcomes with BLASTx analysis. According to the map, to reference analyses of B21+B5 HTS data, 29,574 reads matched with cherry leaf roll virus RNA1 (CLRV) RefSeq NC_015414, and 405 reads matched with CLRV RNA2 RefSeq NC_015415. However, 4 reads matched with cucumber mosaic virus (CMV) RefSeq NC_001440 from B23 HTS data. To confirm the presence of these viruses, virus-specific primers were used to test the total RNA extracted from walnuts. CLRV was detected in sample B21 but not in B5. CMV was not detected in sample B23. Improving nucleic acid extraction methods is recommended to enhance the detection of viral agents using high-throughput sequencing.

Keywords

Niğde İli Ceviz (Juglans spp.) Ağaçlarındaki Viral Etmenlerin Yüksek Kapasiteli Dizileme ile Araştırılması

Öz

Niğde ilinde, Ekim 2023'te, virüs benzeri simptomlar gösteren ceviz ağaçları, viral enfeksiyonları araştırmak için yüksek kapasiteli dizileme kullanılarak analiz edilmiştir. Çift sarmallı RNA (dsRNA) ekstraksiyonu yapılmış, ardından dsRNA'lar cDNA'ya dönüştürülmüştür. Daha sonra metagenomik analizler için MID primerleri ile barkodlama kullanılarak PCR gerçekleştirilmiştir. B13, B22, B23 ve B21+B5 ceviz örneklerinin PCR ürünleri Illumina NovaSeq 6000 dizileme platformu kullanılarak dizilenmiştir. Dizileme verisi Geneious Prime ve CLC Workbench kullanılarak biyoinformatik analize tabi tutulmuştur. Bu karşılaştırmada, CLC Workbench'te daha fazla contig oluşturulmuş ve BLASTx analizi ile daha doğru sonuçlar elde edilmiştir. B21+B5 HTS verisinin referansa göre haritalama-analizlerine göre, 29.574 okuma cherry leaf roll virüsü RNA1 (CLRV) RefSeq NC_015414 ile ve 405 okuma CLRV RNA2 RefSeq NC_015415 ile eşleşmiştir. Ancak, B23 HTS verisindeki 4 okuma cucumber mosaic virus (CMV) RefSeq NC_001440 ile eşleşmiştir. Bu virüslerin varlığını doğrulamak amacıyla, cevizlerden ekstrakte edilen total RNA'yı test etmek için virüse özgü primerler kullanılmıştır. CLRV, B21 örneğinde tespit edilmiş ancak B5'te tespit edilmemiştir. CMV, B23 örneğinde tespit edilmemiştir. Yüksek kapasiteli dizileme kullanılarak viral ajanların tespitini arttırmak için nükleik asit ekstraksiyon yöntemlerinin iyileştirilmesi önerilmektedir.

Anahtar Kelimeler

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Details

Primary Language

English

Subjects

Phytopathology

Journal Section

Research Article

Authors

Bahar Makbule Temeltürk ^*
0009-0003-2834-0947
Türkiye

Serkan Önder
0000-0001-8954-8707
Türkiye

Çiğdem Ulubaş Serçe
0000-0001-5337-5883
Türkiye

Early Pub Date

May 1, 2025

Publication Date

June 9, 2025

Submission Date

January 13, 2025

Acceptance Date

February 26, 2025

Published in Issue

Year 2025 Volume: 28 Number: 3

DOI

https://doi.org/10.18016/ksutarimdoga.vi.1618751

IZ

https://izlik.org/JA74HK92BA

Cite

RIS / Bibtex

APA

Temeltürk, B. M., Önder, S., & Ulubaş Serçe, Ç. (2025). Investigation of Viral Agents in Walnut (Juglans spp.) Trees by High Throughput Sequencing from Niğde province, Türkiye. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Ve Doğa Dergisi, 28(3), 820-829. https://doi.org/10.18016/ksutarimdoga.vi.1618751

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