Complete Genome sequence of a new variant of jujube mosaic-associated virus isolated from jujube (Ziziphus jujuba Mill.) grown at Aksu in Xinjiang of China

This work reports the discovery and the complete genome sequence of a novel member of the genus Badnavirus in the family Caulimoviridae from a Chinese jujube tree of known variety which grown in Aksu, Xinjiang, China. The symptoms of jujube leaves infected by the virus showed mosaic and malformation, and round chlorotic spots on infected fruits. The genome of this virus is a circular double-stranded DNA, which has the length of 7086 nt and has a genome structure similar to the one reported for jujube mosaic-associated virus (JuMaV), with ve open reading frames (ORFs). High nucleotide and amino acid sequence similarity were seen between JuMaV and the new virus, and the nucleotide (NT) difference of the genome was greater than 20% in the RT/ Rnase H coding region of ORF3. Consequently, the virus was identied a new variant of JuMaV, and propose the name as jujube mosaic-associated virus A (JuMaVA).

many times in China. However, pathogenic mechanism of JuMD needs further research and exploration.
Two complete sequences of JuMD can be obtained from the database, jujube mosaic-associated virus (JuMaV, KX852476.1) and jujube associated Badnavirus (JuAB, MN274946.1), respectively. Although sequence analysis of JuMaV has been reported, diseased leaves were collected from Chinese jujube trees grown in Beijing, China [14], there is no information on JuMD in other regions and jujube variety.
In July 2020, leaf and fruit samples were collected from other single plant of Z. jujuba cv. 'Huizao' (sample IDs: HZ, AKS-6) growing areas in Aksu, Xinjiang Uygur Autonomous Region, China. These plants showed symptoms of mosaic and malformation on young leaves (Fig. 1A, left panel), and round chlorotic spots on infected fruits (Fig. 1A, right panel). All leaf and fruit samples were mixed into one pool, respectively, from which total RNA was extracted using a TransZol Up Plus RNA Kit (Transgen) for Small RNA and RNA Sequencing analysis.
A cDNA library of sRNAs library was constructed and sequenced on an Illumina HiSeq XTen sequencing machine (Illumina) with a paired-end 150 bp setup [14]. A total of 179 contigs length sequence between 34 to 448 nt were found in leaf and fruit samples, which had high sequence identity ranging from 46.3% to 94.44% with several badnaviruses. Therefore, we concluded that the virus was a badna-like virus found in mosaic-diseased jujube trees.
Total DNA was extracted from obviously symptomatic leaves and fruits using DNAsecure Plant Kit. In order to identity the virus, two degenerate primers pair BADNA-FP/RP were used to screen sequence of badnavirus from the total DNA [15], designed to amplify 529 bp sequences in the reverse transcriptase/RNase H region has been used for taxonomic purposes within this genus [1]. The PCR of leaves and fruits samples both yielded an amplicon of approximately 500 bp were analysed by gel electrophoresis in 1.5% (w/v) agarose gels. The amplicons were cloned into the pEASY ® -T1 Cloning vector, and sequenced. BLAST analysis of the sequencing results revealed that the 528-bp PCR fragment shared maximum sequence identity of 81.8% with the corresponding regions of Jujube mosaicassociated virus (JuMaV, KX852476.1) belong to the genus badnaviruses. Based on the JuMaV alignment, 5 primers were designed spanning the whole genome and used to obtain the full-length genome sequence of this badnavirus (Table 1). PCR ampli cations were performed in 25-µl reaction mixtures containing 1 µl of DNA template, 13µl 2×Es Taq MasterMix by the manufacturer (CWBIO), 1 mM each primer, 9µl of ddH 2 O. PCR cycling was as follows: initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a nal extension at 72°C for 10 min.
Amplicons were separated by electrophoresis in 1.4% agarose gels puri ed by EasyPure ® Quick Gel Extraction Kit, cloned into the pEASY ® -T1 Cloning vector and transformation of E. coli DH5a, positive clones were obtained and sequenced by Sangon Biotech (Shanghai) Co., Ltd. In all cases, at least three independent clones in both directions were sequenced to determine the exact sequence of the presumed full-length banavirus genome. Genome and conserved domains analysis, genome structures of JuMaVA were representative of typical members of the genus Badnaviruses (Fig. 1B). Several highly conserved motifs were found in the JuMaVA genomes, which had been reported in well-characterized badnavirus genomes [16,17].The JuMaVA genomes contain a tRNA Met binding site (TGGTATCAGAGC [1][2][3][4][5][6][7][8][9][10][11][12]  Phylogenetic trees were generated, which based on full nucleotide sequences of JuMaVA and nucleotide sequences of RT-RNase H region with neighbour-joining (NJ) method using Mega Version7.0 to determine the taxonomic position of JuMaVA. RT/ RNase H sequences were selected for analysis because they play a critical role in viral replication, therefore are subject to more stringent variability constraints. The fullgenome phylogenetic tree shows that JuMaV is in a group with JuMaV (Fig. 1C). The phylogenic tree based on the RT/RNase H nucleotide sequences indicated that JuMaVA clusters with JuMaV (Fig. 1D). The presence of these hallmark features further support the identi cation of JuMaVA as badnaviruses.
Moreover, JuMaVA shares nucleotide sequence identity 99.5% with JuMaV in full sequence and 74.25% in RT-RNase H region. Based on the difference of the genome is greater than 20% in RT-RNase H region, we propose that JuMaVA as a new variant of JuMaV is more accurate.

Conclusion
In conclusion, the rst complete genome sequence of jujube mosaic virus was identi ed isolated from Chinese Jujube trees in Aksu, Xinjiang, China. Not only did we isolate the virus from diseased leaves, but we also isolated the virus from the symptomatic jujube fruit for the rst time. Based on the analysis of full-length genome sequences, the virus was identifed as the closest relative of JuMaV belonging to a member of the badnavirus species in the family Caulimoviridae. We propose this new member of the genus Badnavirus to be designated "Jujube mosaic-associated virus A" (JuMaVA), which is a new variant of JuMaV infecting jujube (Ziziphus jujuba Mill.) grown at Aksu in Xinjiang of China. The work con rmed that the transmission of the virus is widespread and variable. Therefore, we should pay more attention to