Van İlinde Yoncada Saptanan Yonca mozaik virüs (AMV) İzolatlarının Kılıf Protein Genomunun Moleküler Karakterizasyonu

Van ilinde yetiştiriciliği yapılan yonca (Medicago sativa L.) bitkilerinde 2019 yılında virüslerin oluşturduğu simptomlara benzer belirtiler görülmüştür. Gözlenen belirtiler arasında cüceleşme, yapraklarda mozaik deseni ve sararma, rozetlenme ve küçük yaprak oluşumu yer almaktadır. Simptom gösteren ve göstermeyen bitkilerden toplanan 19 yonca yaprak örneğine, virüs genomunu tespit edebilmek için uygun primer çiftleri ile RT-PCR testi uygulanmıştır. Yonca yaprağı örneklerinden şiddetli belirtiye sahip altısı beklenen büyüklükte 700 bp DNA band oluşturmuştur. Bunlar arasından rastgele seçilen ikisi bir plazmid vektörüne klonlanmıştır. Elde edilen rekombinant plazmidler her iki yönde dizilenmiştir. Dizi analizi sonuçlarına göre infekteli yoncalardaki virüsün Yonca mozaik virüs olduğu açığa çıkarılmıştır. Dizi bilgileri MT210179 ve MT210178 erişim numaraları ile gen bankasına yüklenmiştir ve sırasıyla Alakoy Y9 ve Alakoy Y1 olarak isimlendirilmiştir. Her iki dizi için gen bankasına kayıtlı 16 AMV dizisi ile oluşturulan filogenetik ağaca göre, her iki izolat da nükleotit düzeyinde ABD, Brezilya ve Puglia izolatları ile en yüksek benzerlik ve Güney Kore izolatı ile en düşük benzerlik oranı göstermiştir. Ayrıca her iki dizinin birbirleri arasında, 7 nükleotit değişikliği ile %98.45 oranında nükleotit benzerliği gösterdiği ortaya çıkarılmıştır. Gerçekleştirilen litaratür tarama çalışmalarına göre, bu çalışma Türkiye'nin Van ilinde yetiştirilen yonca bitkilerindeki Yonca mozaik virüsü (AMV)’ nün ilk raporu ve moleküler analizidir.

Anahtar Kelimeler:

Dizileme, filogenetik analiz, RT-PCR, karakterizasyon, yonca

Molecular Characterization of the Coat Protein Genome of Alfalfa Mosaic Virus (AMV) Isolates from Alfalfa in Van Province

Virus-like symptoms were observed in alfalfa plants (Medicago sativa L.) grown in Van province of Turkey in 2019. The symptoms observed were dwarfing, mosaic pattern and yellowing of leaves, rosetting, and decreasing in leaf sizes. Genomic RNA of 19 symptomatic and non-symptomatic alfalfa leaves were extracted for the detection of virus RNA using virus-specific primer pair by RT-PCR. Of the 19 alfalfa leaf specimens, the six yielded the expected 700 bp DNA band in severely symptomatic alfalfa specimens. Two of them randomly selected were inserted into a plasmid vector. Obtained recombinant plasmids were sequenced in both directions. According to the results of the sequence analysis, it was revealed that the virus in infected alfalfa was the Alfalfa mosaic virus. The sequence data were recorded into the GenBank with access numbers MT210179 and MT210178 and denominated as Alakoy Y9 and Alakoy Y1 isolates, respectively. According to the phylogenetic tree created with 16 AMV sequences registered in the GeneBank for both sequences, the AMV CP gene sequence indicated the highest similarity with USA (JN256023), Brazil (FJ858265), and Puglia (Y09110) isolate and the lowest with Korea isolate (KY348844), at the nucleotide level. It was also revealed that both sequences show 98.45% nucleotide similarity with 7 nucleotide exchanges. According to the literature research results, this is the first report and molecular analysis of Alfalfa mosaic virus (AMV) in Medicago sativa L. in Van province of Turkey.

Keywords:

Alfalfa, RT-PCR, characterisation, sequencing, phylogenetic analysis,

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