JIA-2019-11

2665 HU Qian-qian et al. Journal of Integrative Agriculture 2019, 18(11): 2664–2667 while ORFs Ib, VII and VIII are nonessential for replication and systemic infection, their functions remain unknown (Hasegawa et al . 1989; Takemoto and Hibi 2001, 2005). So far, SbCMV has only been reported in Japan and only two full genome sequences were found in the GenBank (Hasegawa et al . 1989). 2. Materials and methods 2.1. Virus sources, RNA extraction and deep sequencing From 2013 to 2015, legume plants showing leaf curling, leaf mottle, mosaic and chlorotic symptoms were collected from fields in Jiangxi and Zhejiang provinces of China. Total RNA was extracted from fresh leaf tissue using TRIzol reagent according to the manufacturers’ instructions (Invitrogen, USA) and sequenced on the Illumina Hiseq 2000 Solexa platform by Beijing Novogene Biological Technology Co., Ltd., China (www.novogene0.bioon.com.cn/ ). 2.2. DNA extraction, genome cloning and sequencing Total DNA was extracted from symptomatic leaves using a cetyltrimethylammonium bromide (CTAB)-based method as described by Zhong et al . (2017). PCR amplifications with the specific primers were performed as instructed by the manufacturer. PCR products were recovered from agarose gel with AxyPrep DNA Gel Extraction Kit (Axygen Bioscience, Shanghai, China) and cloned into pMD 18-T vector (TaKaRa Biotechnology, Dalian, China) and sequenced by conventional Sanger sequencing. The primers used for viral amplification were listed in Appendix A. 2.3. Sequence and phylogenetic analyses Sequence data were assembled and analysed by SeqMan (Lasergene Package, version 7.1.0). Phylogenetic dendrogram tree was constructed by the Clustal W and neighbor-joining method with 1 000 bootstrap replicates using the MEGA version 6 Software. The accession numbers of viral sequences used for comparison were listed as follows: SbCMV-JA (X15828, E02829), BRRSV (AF404509), PCSV (U13988), CmYLCV (AF364175), Cauliflower mosaic virus (CaMV, NC001497), Cassava vein mosaic virus (CsVMV, NC_001648), Commelina yellow mottle virus (ComYMV, X52938), Petunia vein clearing virus (PVCV, U95208) and Rice tungro bacilliform virus (RTBV, X57924). 3. Results 3.1. Identification of SbCMV by deep sequencing In 2013, 17 legume samples including soybean, cowpea and kidney bean with leaf curling, leaf mottle, mosaic and chlorotic symptoms from fields in Jiangxi and Zhejiang provinces of China were chosen for deep sequencing. A total of 27 613 568 clear sRNA reads were obtained and the 11 contigs assembled from 4324 reads were 92–99% identical with the genome of SbCMV. Based on the sequenced contigs, primers SbCMV-F1 and SbCMV-R1 were designed to amplify possible SbCMV in the 17 legume samples by PCR, and a 400-bp specific product was detected in soybean sample NC113 from Nanchang City of Jiangxi Province, which was 94% identical with that of SbCMV. Then three PCRs were performed using the primer pairs SbCMV-F2/ R2, SbCMV-F3/R3 and SbCMV-F4/R4 to yield the 2.5, 5.4 and 1.6 kb bands, respectively, and the full-length genome of NC113 was assembled with these three overlapped amplified fragments. 3.2. Genomic organization of SbCMV-NC113 The complete nucleotide sequence of NC113 was determined to be 8 210 nucleotides (nts) (accession no. MH718847) and had a GC content of 33.58%. Nucleotides were numbered starting from the 5´ nucleotide of the minus-strand primer-binding site (5´-TGGTATCAGAGC-3´), which was complementary to the host cytosolic initiator methionine tRNA for synthesizing the minus-strand DNA and was highly conserved in all members of the Caulimoviridae family. NC113 showed the typical SbCMV genomic organization and encoded nine putative ORFs (ORFs Ia, Ib and II–VIII) with sizes similar to those of SbCMV-JA reported in Japan (accession no. X15828), and contained a large IR of about 537 nts located at nt 5976–6512 between ORFs VI and VII (Table 1). The overall nucleotide sequence of NC113 shared the highest similarity (91.7%) with that of SbCMV-JA, and higher identities with members of Soymovirus (BRRSV, 53.8%; PCSV, 48.7%; CmYLCV, 47.6%) than with all other Caulimoviridae members (less than 40.3%), suggesting NC113 was an isolate of SbCMV. This is the first identification of Soybean chlorotic mottle virus infecting soybean in China. Compared with SbCMV-JA (8 178 nts), the full sequence of SbCMV-NC113 (8 210 nts) was a little longer due to the insertion of a total of 42 nts in the IR, and the IR sequence identity between themwas 85.0% (Table 1). There was a TATA box sequence (TATAAATA) at nt 6144– 6 151 and a TATA-like box (TAATAAA) at nt 6 041–6 047 in