CLONING AND SEQUENCING OF THE COAT PROTEIN GENE OF A CHINESE ISOLATE OF BARLEY MILD MOSAIC VIRUS
TAO ZHENG1, JIANPING CHEN1, YE CHEN1, JF ANTONIW2 and MJ ADAMS2
1Virology Laboratory, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 2IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Background and objectives
Yellow mosaic disease causes serious damage to winter barley crops in Japan, China, Korea and north-west Europe. The agent was first described as barley yellow mosaic virus (BaYMV) but it later became evident that another virus, barley mild mosaic virus (BaMMV), could also be involved, either alone or in association with BaYMV. Both viruses have similar particle morphology and symptomatology, and are transmitted by the fungus Polymyxa graminis. However, they are serologically distinct and differ significantly at the molecular level. Both viruses have a bipartite genome comprising two 3'-polyadenylated ssRNA molecules of 7.3-7.6 kb (RNA1) and 3.5-3.6 kb (RNA2). The complete nucleotide sequences of European and Japanese isolates of both viruses have been published and indicate that each RNA is translated into a polyprotein which is subsequently processed into functional products. The coat protein is at the C-terminus of the RNAI polyprotein. Recently, BaMMV was detected by ELISA in China  and we now present the sequence of its coat protein gene.
Materials and methods
Barley leaves with yellow mosaic symptoms were collected from a field in Rudong county, Jiangsu province, China. ELISA and ISEM using antisera against UK BaYMV or BaMMV indicated that both BaYMV and BaMMV were present. Total plant RNA was extracted from these infected leaves and cDNA of part of BaMMV RNA was synthesised using a BaMMV specific primer ZW2 (5'-GCATGAGAGATCTACCGG-3') complementary to the sequence downstream of the coat protein gene of UK BaMMV. PCR was carried out using primer ZW2 in combination with ZW1 (5'-ACAGAGCACGAGGAGGAA-3'), which matches the sequence upstream of the UK BaMMV coat protein gene. A unique 890-bp fragment was amplified and cloned into pUCI9 at the SmaI site and sequencing was done with a USB sequencing kit according to the supplier's procedure. Sequence analysis was done using the Wisconsin GCG package.
Results and conclusions
The coat protein of the Chinese isolate of BaMMV was identical in length (753 nucleotides; 251 amino acids) to those of isolates from Japan, Korea, Germany, France and the UK. It was most closely related to the Korean (D83410) and Japanese Nal (D83408) isolates (respectively 95.2 and 94.4% nucleotide identity; 97.6 and 96.2% amino acid identity). Using the GCG Pileup program, the available BaMMV coat protein sequences fell into three distinct groups: (i) China, Korea, Japan (Nal); (ii) Japan (Kal: D10949) and German Q(69204); (iii) UK 10973; Y10974) and French (L49381).
The results confirm that the virus detected in China is indeed BaMMV. Although BaYMV is the more important mosaic virus of barley in East Asia, the results suggest that an Asian strain of BaMMV has also been long established.
1. Chen J, Adams MJ, Zhu F et al., 1996. Plant Pathology 45, 1117-1125.