Complete genomic sequence and recombination analysis of wheat yellow mosaic virus isolate from Zhouzhi in China

Wheat yellow mosaic virus (WYMV) is the causal agent of wheat yellow mosaic disease in China. WYMV was detected in wheat sample collected from Zhouzhi of Shanxi province. The nearly complete genomic sequence of Zhouzhi isolate (WYMV-ZZ) was determined; it was compared with six complete sequences of WYMV isolates (five Chinese isolates and one Japanese isolate). WYMV-ZZ and the other six different WYMV isolates shared 96.6 to 97.7% and 95.1 to 98.2% nucleotide sequence identity for RNA1 and RNA2, respectively; at the amino acid level, WYMV-ZZ had 94.1 to 98.2% identity for RNA1 and 94.1 to 96.7% identity for RNA2, respectively, with the other six isolates. Phylogenetic analysis showed that the NIa-VPg region can separate the Chinese isolates from Japanese isolate. Based on the recombinant analysis, there were three possible recombination events; one event was located in RNA1 CI region of WYMV-ZZ with a RDP P-value of 8.526×10 -06 . This work advanced our understanding of the WYMV molecular variation and was helpful to study the disease spread.


INTRODUCTION
Wheat yellow mosaic virus (WYMV), is the causal agent of wheat yellow mosaic disease of wheat in China and Japan, belongs to the genus Bymovirus within the family Potyviridae and is a soil-borne pathogen, it is transmitted by the fungus-like organism Polymyxa graminis (Sawada, 1927).In China, the disease was found in Sichuan province in the 1960s (Tao et al., 1980) and spread gradually to the middle and lower valleys of the Yangtze and Huai Rivers (Li et al., 1997;Chen, 1999).Wheat yellow mosaic virus causes typical symptoms including mosaic, yellowing, dwarfing, stunting or excessive tillering, and subsequently decreasing yield.Under lowtemperature conditions in the field, WYMV infects wheat.When spring comes, the infected wheat shows light green, oval-or spindle-shape spots; the temperature back to 10°C, the infected leaves show yellow mosaic symptom with the disease spots expand and emerge.Finally, WYMV causes serious damage as a result of *Corresponding author.E-mail: hanchenggui@cau.edu.cn.Fax: +86-010-62813758.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License yield losses (Wang et al., 2015).

Sample
Wheat sample was collected from Zhouzhi, Shanxi province of China in 2008.

Total RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR) and sequencing
Total RNA from wheat leaves was extracted by LiCl precipitating method (Zhang et al., 2011).Primer VP-1M was used for reverse transcription (RT) reaction and primer pair VP-1P/VP-1M was used to amplify a 704 bp fragment which was the VPg region in RNA1; primer ut-1M was used for RT reaction and primer pair ut-1P/ut-1M was used to amplify a 880 bp fragment which was the 3-termino-UTR in RNA2 (Ohto and Sakai, 2005).The purified bands were cloned into pMD18-T vector and 2-3 clones were sequenced by companies (Introvigen and BiMad).The sequence fragments were combined together by DNAMAN 7.0.

Western blotting
The wheat leaves were grinded by liquid nitrogen, added 2×SDS protein buffer, blended and incubated at 100°C for 5 min, then put the samples on the ice for 5 min, and centrifuged 12,000 rpm for 10 min; the supernatant was carried onto the SDS polyacrylamide gel electrophoresis.After electrophoresis, the sample was transferred to Hybond-C membrane, and the membrane was incubated with TBST buffer (20 mM Tris, 137 mM NaCl, 0.3% Tween20, pH 7.6) containing 5% skimmed milk powder for 2 h at 37°C and added antiserum of WYMV-CP which was prepared by Yan-hong Han storing in my lab to incubate for 1 h at 37°C.Blot was rinsed by TBST for 3 times and incubated for 1 h with anti-goat IgG diluted 1:10,000.After washing in TBST, blot was visualized by NBT and BCIP (Han et al., 2002).

Phylogenetic analysis
To better understand the relationship of WYMV-ZZ and other six WYMV isolates, the full-length sequence alignments and phylogenetic analysis of nucleotide and amino acid were conducted.Phylogenetic trees were constructed for by the neighbor-joining method and visualized using MEGA X (Molecular Evolutionary Genetics Analysis version X) with 1000 bootstraps replicate (https://www.megasoftware.net)(Kumar et al., 2018).

Recombination analysis
Recombination of seven WYMV isolates was constructed by RDP4.97 (Recombination Detection Program version 4.97) (Martin et al., 2015).Various recombination detection methods were used to analyze putative recombinants and recombination breakpoints, including the programs RDP, GENECONV, BOOTSCAN, MAXCHI, SISCAN and 3SEQ.The recombination events which were surveyed by at least five different methods could be received (Zhou et al., 2012).

Detection of WYMV-ZZ by RT-PCR and Western blotting
WYMV-ZZ was detected from wheat sample of Zhouzhi, Shanxi province using RT-PCR and Western blotting (Figure 1).

Complete genomic sequence of WYMV-ZZ and its comparison with other six isolates
The genomic RNA sequence of WYMV-ZZ was obtained from the wheat sample by amplification of four overlapping cDNA fragments for RNA1 and two overlapping cDNA fragments for RNA2 using the primer pairs WY1001F/WY11920R, WY11858F/WY13832R, WY13578F/WY15446R, WY15378F/HC511-BHR for RNA1, and WY2001F/WY22012R, WY21927F/HC511-  BHR for RNA2 (Table 1).These primers were derived from the conserved region of six known isolates.A nearly complete nucleotide sequence of WYMV was determined, apart from short regions where the primers annealed at the 5'-and 3'-terminus.The full-length sequence of WYMV was submitted to GenBank with accession number FJ261765 for RNA1 and FJ361768 for RNA2 (Table 2).

Phylogenetic analysis of seven different isolates
To better understand the relationship between WYMV-ZZ and six other isolates, the phylogenetic analysis of seven different isolates was constructed.Phylogenetic trees were constructed for P3, 7K, CI, 14K, NIa-VPg, NIa-Pro, CP, and full-length of RNA1, and for P1, P2, and fulllength of RNA2 by the neighbor-joining method and visualized using MEGA (version X) with 1000 bootstrap replicates.The results showed that P3, CI, 14K, NIa-VPg, NIa-Pro, NIb and CP of WYMV-ZZ were more closely related to WYMV-YA, 7K of WYMV-ZZ was close to WYMV-XQ; and that P1 and P2 of WYMV-ZZ were close to WYMV-ZMD (data not shown).The full-length of WYMV-ZZ was close to WYMV-YA for RNA1 and was close to WYMV-ZMD for RNA2 (Figure 2A and B).The phylogenetic trees generated based on the NIa-VPg region of RNA2 showed that this region clustered together with the other five Chinese isolates, while WYMV-JPN formed a distinct branch (Figure 2C and D).

Recombination analysis
The seven sequences of WYMV isolates were processed and examined for recombination at the same time.The major parent, minor parent, the event and the corresponding P-value of four recombination events are as shown in Figure 3 and Table 4.The most possible one recombination event of WYMV-ZZ, which is located in 2,598 to 4,019 nt of RNA1 CI region, may recombined with unknown major parent (WYMV-JPN) and minor parent (WYMV-HC) with a RDP P-value of 8.526×10 -06 . The second recombination event of WYMV-HC, which is located in 564 to 894 nt of RNA2 P1, may recombined with major parent (WYMV-ZZ) and minor parent (WYMV-YA) with a RDP P-value of 5.444×10 -05 . The third recombination event of WYMV-XQ, which is located in 1698 to 3196 nt of RNA P2 and 3′ UTR regions, may recombined with major parent (WYMV-ZMD) and minor parent (WYMV-YA) with a RDP P-value of 3.147×10 -05 . In this study, WYMV was detected in wheat leaves which is collected from Zhouzhi, Shanxi province of China in 2008.The wheat samples were infected with WYMV confirming by both RT-PCR and western blotting.For RT-PCR, targeting the VPg region in RNA1, primer VP-1M was used for reverse transcription reaction and primer pair VP-1P/VP-1M was used to amplify a 704 bp fragment; targeting the 3-termino-UTR in RNA2, primer ut-1M was used for RT reaction and primer pair ut-1P/ut-1M was used to amplify the 880 bp fragment.Using antiserum of WYMV-CP, Western blotting was carried out with the 32 kD positive band (Figure 1B).
Apart from short regions where the primers annealed at the 5'-and 3'-terminus, a nearly complete nucleotide sequence of WYMV-ZZ was determined and given the GenBank accession number FJ361765 for RNA1 and FJ361768 for RNA2, respectively (Table 2).
Based on the nucleotide sequence comparison, WYMV-ZZ shared 96.6 to 97.7% and 95.1 to 98.2% nucleotide sequence identity for RNA1 and RNA2 with the other six isolates (WYMV-HC, WYMV-YA, WYMV-YZ, WYMV-XQ, WYMV-ZMD, WYMV-JPN) infecting wheat in different parts of the world.At the amino acid level, WYMV-ZZ had 94.1 to 98.2% identity for RNA1 and 94.1 to 96.7% identity for RNA2 with other six isolates.The identity for genes of RNA1 and RNA2 were >90% between WYMV-ZZ and six other isolates (Table 3).
To better understand the relationship between WYMV-ZZ and other six isolates, the phylogenetic analysis were carried out using MEGA (version X) with 1000 bootstrap replicates.The results showed that P3, CI, 14K, NIa-VPg, NIa-Pro, NIb and CP of WYMV-ZZ were more closely related to WYMV-YA, 7K of WYMV-ZZ was close to WYMV-XQ; and that P1 and P2 of WYMV-ZZ were close Detected by at least five different methods, three recombination events were received (Zhou et al., 2012).The recombination event detected in RNA1 CI region of WYMV-ZZ, which is located in 2,598-3,344 nt, may be recombined with unknown major parent (WYMV-YZ) and minor parent (WYMV-HC) with a RDP P-value of 8.526×10 -06 .There was no recombination event RNA2 of WYMV-ZZ.Consistent nucleotide and amino acid, and close phylogenetic relationships may imply the molecular evolution of WYMV is a genetic stability progress.

DISCUSSION
The genomic RNA sequence of WYMV-ZZ was determined.Sequence comparison, phylogenetic tree and recombination analysis were performed among WYMV-ZZ and other six known WYMV isolates.Consistent nucleotide and amino acid, and close phylogenetic relationships may imply the molecular evolution of WYMV is a genetic stability progress.
VPg is a multiple function protein, which participates in the genomic replication and interacts with 3-terminal poly-A to achieve similar function with 5-terminal cap structure (Gallie et al., 1995;MURPHY et al., 1996;Ohshima et al., 2007)

CFigure 3 .
Figure 3. Recombination of WYMV analyzed using RDP4.97.A-D: BOOTSCAN plot for the recombinant of WYMV-ZZ within RNA1, of WYMV-HC within RNA2, and of WYMV-YZ within RNA2.The left and the right boundaries of the pink region indicate breakpoint positions.The yellow line is the major parent: minor parent plot, the green line is the recombinant plot; the dotted line indicates the bootstrap cut off value.

Table 1 .
Primers used for RT-PCR and determining full-length sequences.

Table 2 .
GenBank accession numbers of seven WYMV isolates.

Table 3 .
Sequence identity comparison of WYMV-ZZ with other six isolates.