First report of barley virus g in proso millet (Panicum miliaceum) in Korea

Proso millet (Panicum miliaceum L.) is commonly used as an edible cereal crop and its cultivation area is increasing gradually in Korea. From June to August 2015, a nationwide survey was carried out to investigate viral disease infection in proso millet. A total of 64 proso millet leaf samples showing yellowing stripe, necrosis, and mosaic symptoms were collected and saved for future virus testing. All collected leaves were combined into one sample and total RNA was extracted from the pooled sample using an Easy-Spin total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea). High-throughput paired-end RNA sequencing was performed using an Illumina HiSeq2500 sequencer by Theragen Etex Bio Institute (Suwon, Korea) as described previously (Zhao et al. 2016) and raw data were analyzed by SEEDERS Company (Daejeon, Korea). Among the analyzed contigs, 59 viral contigs (51 contigs from Rice stripe virus [RSV] and eight contigs from Barley virus G [BVG]) were obtained. RSV in proso millet and BVG (BVG Gimje isolate) in barley (Hordeum vulgare L.) have been reported in Korea and BVG is considered as a new species in the genus Polerovirus (Yoon et al. 2012; Zhao et al. 2016). To confirm the presence of BVG, total RNA was extracted from 64 individual samples and reverse-transcriptase (RT)-PCR was performed using two specific primer sets (RSV: 5′-GATCTGAGGGGTTCACATAGGACT-3′ and 5′-GATGCGCTGGCTGGTATGTCT-3′; BVG: 5′-GACATCTACTCTTTGAGTTTC-3′ and 5′-GTTCCGAAGAGGAATAAGGA-3′), yielding products of the expected sizes (353 bp for RSV and 581 bp for BVG) in two RSV-positive and seven BVG-positive samples. To confirm the nucleotide sequence of the coat protein (CP) of BVG isolated from proso millet, a primer set (5′-GTGAGTTGCAAGTACTGGAT-3′ and 5′-GTACCCTGCCGAAAGTGTT-3′) was used. A PCR product of the expected size (988 bp) from the seven BVG-positive samples was cloned and sequenced. NCBI BLAST analysis showed that the BVG CP gene isolated from proso millet had the highest nucleotide identity (99%) with the BVG Gimje isolate (GenBank accession no. KT962089) and was deposited in GenBank under accession number LC159487 (BVG Uiseong isolate). This is the first report of BVG infection of proso millet in Korea and the world. Further studies are needed to elucidate the virus characteristics, such as transmission, natural host range, and geographical distribution.