番红花种球中虎眼万年青花叶病毒的分子鉴定与序列分析

doi:10.3864/j.issn.0578-1752.2017.20.019

Abstract

Abstract: 【Objective】The objective of this study is to identify viruses in saffron (Crocus sativus) corms imported from the Netherlands and to prevent the entry of dangerous virus into China. 【Method】Using the Potyviridae universal primers (Sprimer and M4T), the saffron corms samples were detected by reverse transcription polymerase chain reaction (RT-PCR) assay. After cloning and sequencing of the RT-PCR products, the Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Comparison by Log-Expectation (MUSCLE) methods were used for multiple sequence alignment. The maximum likelihood method was used to construct the phylogenetic tree according to the coat protein (CP) nucleotide sequences. 【Result】The about 1.7 kb RT-PCR products were amplified from the samples, indicating a member of Potyviridae virus was present in the samples of saffron corms. A sequence of 1 666 bp segments was obtained from a positive sample (designated 2677-NL) and it contained a portion of nuclear inclusion b gene (NIb), the complete CP and 3′ untranslated region (3′-UTR). This isolate shared the highest nucleotide sequence identity (99.6%) with the SW3.3 isolate of Ornithogalum mosaic virus (OrMV) and shared 99.1% nucleotide sequence identity with the Reference genomic sequences (RefSeq) of OrMV (GenBank accession number: NC_019409). Of those, the CP of the 2677-NL isolate shared 71.5%-77.3% and 65.7%-79.7% nucleotide and amino acid identities, respectively, with those of six OrMV isolates from India (GenBank accession number: JQ686722, JQ686720, JN692498, JN692497, JN692496 and JF682235) whereas 77.9%-99.5% and 82.9%-99.2%, respectively, with the other 32 OrMV isolates. Phylogenetic analysis based on the CP genes showed that the OrMV could be divided into two groups, in which 2677-NL was clustered together with five OrMV isolates from Australia (GenBank accession number: JQ807995, JQ807996, NC_019409, AF185964 and AF185965), suggesting these OrMV were highly homologous in the phylogenetic relationship. 【Conclusion】According to the species demarcation criteria of the family Potyviridae on the International Committee on Taxonomy (ICTV), the 2677-NL was an isolate of OrMV, and it showed that the saffron corms sample was infected by OrMV. this is the first report on OrMV infecting C. sativus in the world.

Key words: Ornithogalum mosaic virus, Crocus sativus, RT-PCR, new host

LIAO FuRong, LIN WuZhen, CHEN XiHong, CHEN Qing, CHEN HongYun, HUANG PengYing, FANG ZhiPeng, WU Yuan, SHEN JianGuo, LIN ShiMing. Molecular identification and sequence analysis of Ornithogalum mosaic virus in saffron (Crocus sativus) corms[J].Scientia Agricultura Sinica, 2017, 50(20): 4046-4054.

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Molecular identification and sequence analysis of Ornithogalum mosaic virus in saffron (Crocus sativus) corms

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