Plant Pathol J > Volume 33(5); 2017 > Article
Jo, Choi, Bae, Kim, Kim, Lee, Cho, and Kim: De novo Genome Assembly and Single Nucleotide Variations for Soybean Mosaic Virus Using Soybean Seed Transcriptome Data

Abstract

Soybean is the most important legume crop in the world. Several diseases in soybean lead to serious yield losses in major soybean-producing countries. Moreover, soybean can be infected by diverse viruses. Recently, we carried out a large-scale screening to identify viruses infecting soybean using available soybean transcriptome data. Of the screened transcriptomes, a soybean transcriptome for soybean seed development analysis contains several virus-associated sequences. In this study, we identified five viruses, including soybean mosaic virus (SMV), infecting soybean by de novo transcriptome assembly followed by blast search. We assembled a nearly complete consensus genome sequence of SMV China using transcriptome data. Based on phylogenetic analysis, the consensus genome sequence of SMV China was closely related to SMV isolates from South Korea. We examined single nucleotide variations (SNVs) for SMVs in the soybean seed transcriptome revealing 780 SNVs, which were evenly distributed on the SMV genome. Four SNVs, C-U, U-C, A-G, and G-A, were frequently identified. This result demonstrated the quasispecies variation of the SMV genome. Taken together, this study carried out bioinformatics analyses to identify viruses using soybean transcriptome data. In addition, we demonstrated the application of soybean transcriptome data for virus genome assembly and SNV analysis.

Introduction

Soybean (Glycine max (L.) Merr.) is the most important legume crop, representing 50% of the global legume crop area and 68% of global legume production (Herridge et al., 2008). Soybean is consumed as health food, providing a rich source of proteins, and as well as vegetative oil production (Messina, 1999; Pimentel and Patzek, 2005). Moreover, soybean plays an important role for dinitrogen (N2) fixation, which is an important natural process (Herridge et al., 2008).
Several diseases in soybean, such as cyst, brown spot, charcoal rot, and sclerotinia stem rot, lead to yield losses in major soybean-producing countries (Wrather et al., 2001). In addition, soybean can be infected by diverse viruses. Although a small numbers of viruses infecting soybean cause serious economic problems in soybean production, it is always important to control and to manage viral diseases in soybeans (Hill and Whitham, 2014). The best known soybean virus is Soybean mosaic virus (SMV), a member of the family Potyviridae, causing soybean mosaic disease. In addition, bean pod mottle virus (BPMV), soybean vein necrosis virus, tobacco ringspot virus, soybean dwarf virus, peanut mottle virus, peanut stunt virus, and alfalfa mosaic virus are important viruses infecting soybeans (Hill and Whitham, 2014).
Many plant viruses have been identified based on viral disease symptoms and several detection methods. However, virus infection in plants does not always cause disease symptoms, and many plants showing viral disease symptoms are very often co-infected by different viruses. Recent advances in next generation sequencing (NGS) technology lead to identification of numerous known as well as novel viruses by means of metagenomics (Barba et al., 2014; Massart et al., 2014). Not only NGS data for virus detection but also many plant transcriptome data contain virus sequences, which might be amplified along with infected host transcripts (Burger and Maree, 2015; Jo et al., 2016). The identification of virus sequences in the plant transcriptome is no longer surprising, because most plant viruses are RNA viruses and many of them carry poly(A) tail, which is easily amplified by oligo d(T) primers for cDNA synthesis.
Recently, we carried out a large-scale screening to identify viruses infecting soybean in the world using available soybean transcriptome data. Of them, we found that a soybean transcriptome for soybean seed development analysis contains many virus sequences. In this study, we conducted a bioinformatics analyses for virus identification, virus genome assembly, phylogenetic analysis, and single nucleotide variations of the SMV.

Materials and Methods

Plant materials, library preparation, and next generation sequencing

The plant material used for RNA-Seq was soybean cultivar Heinong44. Plants were grown in the experimental station in Beijing from May to August according to the previous study (Song et al., 2013). Total RNAs were extracted from seeds at six different developmental stages, which were classified according to the seed weight. The cDNA was synthesized using poly(A)-containing RNAs. A single RNA-Seq library was constructed and sequenced by single-end sequencing using the Illumina HiSeq 2000 system. The raw data is available in the SRA database (http://www.ncbi.nlm.nih.gov/sra/SRR1777405).

Raw data processing and de novo transcriptome assembly

All bioinformatics analyses were performed in the Linux (Linux Mint version 17)-installed workstation (four 16-core CPUs and 256 GB ram). We downloaded the raw data from the SRA database using the SRA toolkit (Leinonen et al., 2011). The raw SRA data were converted to FASTQ files using the SRA toolkit. For the de novo assembly of transcriptomes, we used Trinity version 2.0.6 (Haas et al., 2013). De novo transcriptome assembly was performed according to the manuals provided by developers with default parameters.

Identification of viruses and sequence alignment

To identify virus-associated contigs, we conducted blast search using standalone BLAST version 2.1.19 installed in the Linux system (Madden, 2013). All assembled contigs were subjected to MEGABLAST search, which is optimized for highly similar sequences, against complete reference sequences for viruses and viroids (http://www.ncbi.nlm.nih.gov/genome/viruses/) with E value 1e-5 as a cutoff. In addition, all raw data were converted to FASTA files using the SRA toolkit and subjected to a MEGABLAST search against the viral reference database with E value 1e-5 as a cutoff. We used the Burrows-Wheeler Aligner (BWA) software for sequence alignment on the reference virus genome with default parameters (Li and Durbin, 2009).

De novo assembly of SMV genomes

The 79 SMV-associated contigs identified by the BLAST were retrieved by the BLASTCMD program in the standalone BLAST system. To assemble SMV genomes, the identified viral contigs were aligned against the SMV reference genome (NC_002634.1) using ClustalW implemented in the MEGA6 program (Tamura et al., 2013) The nearly complete consensus genome of SMV was manually obtained. Raw data were again aligned on the assembled consensus SMV genome to confirm sequences by BWA. The poly(A) tail at the 3′ end of the assembled SMV genome was removed. We obtained a nearly complete consensus genome for SMV China (accession number NC_002634.1) from soybean transcriptome.

Identification of SNVs in soybean transcriptome

In order to analyze SNVs of SMV China in the soybean transcriptome, the raw data were aligned on the consensus genome of SMV China using the BWA program with default parameters. The aligned SAM files by BWA were converted into BAM files by SAMtools (Li et al., 2009). For SNV calling, we sorted the BAM files and then generated the VCF file format using mpileup (Danecek et al., 2011). BCFtools implemented in SAMtools was finally used to call SNVs. The positions of identified SNVs on the SMV genome were visualized by the Tablet program (Milne et al., 2010).

Construction of phylogenetic trees

In order to reveal phylogenetic relationships of the obtained consensus genome for SMV China with known SMV isolates, we generated three phylogenetic trees. The complete SMV isolate China genome sequence as well as two polyprotein sequences were blasted against NCBI nucleotide and non-redundant protein databases. Best-matched sequences were retrieved for the construction of phylogenetic tree. The obtained sequences were aligned by the ClustalW program with default parameters. After alignment, we deleted unnecessary sequences. The manually edited aligned sequences were subjected to construction of a phylogenetic tree using the MEGA6 program. The phylogenetic tree was constructed by the neighbor-joining method, with 1,000 bootstrap replicates.

Results

De novo soybean transcriptome assembly and identification of viruses in the soybean seeds

We screened available soybean transcriptome data deposited in NCBI’s Sequence Read Archive (SRA) database in order to identify viruses infecting soybean. Of screened soybean transcriptomes, a transcriptome conducting a gene expression profile during soybean seed development contains several virus-associated sequences (accession number SRR1777405) (Song et al., 2013). In order to identify virus-associated contigs, we de novo assembled the transcriptome of soybean using Trinity program, resulting in 116,108 transcripts (contigs) with 710 bp for contig N50 (Table 1). Next, we blasted 116,108 transcripts against the viral reference database. After removing redundant sequences and endogenous viral sequences, we identified 83 contigs-associated with viruses (Table 2). Most contigs (79 contigs) were associated with SMV. The lengths of SMV-associated contigs ranged from 224 to 3,636 nt (Fig. 1A). Four contigs were associated with BPMV, lettuce infectious yellow virus (LICV), lettuce chlorosis virus (LCV), and cucumber mosaic virus (CMV), respectively. The lengths of contigs associated with the four viruses ranged from 232 nt (LCV RNA2) to 1,015 nt (bean common mosaic virus) (Fig. 1A). Other than a contig-associated with LICV (1E-08), virus-associated contigs display reliable E values indicating significance of blast results (Table 2).

De novo genome assembly of SMV from a soybean transcriptome

Of identified viruses, SMV was severely infected in the soybean seeds. Fortunately, 79 contigs associated with SMV mostly covered the SMV reference genome (Table 2). A total of 79 contigs associated with SMV were mapped on the SMV reference genome (accession number NC_002634.1) (Eggenberger et al., 1989) (Fig. 1B). After sequence alignment followed by manual modification, we assembled a nearly complete consensus genome of SMV referred as SMV China (Fig. 1C). The SMV China is composed of 9,507 nucleotides (nt) encoding two proteins such as GP1 and GP2. GP1 encodes a polyprotein (nt 54 to 9,254) which is further cleaved into ten mature proteins such as P1 (P1 proteinase), HC-Pro (helper component proteinase), P3 (P3 protein), 6K1 (6K1 protein), CI (cylindrical inclusion), 6K2, NIa-VPg (Nuclear inclusion protein a-genome linked viral protein), NIa-Pro, NIb (nuclear inclusion protein b), and coat protein (CP) while GP2 encodes PIPO (pretty interesting potyviridae ORF) protein (nt 2,804 to 3,031) (Fig. 1C).

Phylogenetic relationships of the SMV isolate China

In order to find genetic relationships of the assembled SMV China with known SMV isolates, we constructed phylogenetic trees. The phylogenetic tree using SMV complete genome sequences showed two groups of SMV isolates (Fig. 2A). The SMV China belongs to group B along with two SMV isolates from South Korea. Using polyprotein sequences, the SMV China in group C was distantly related with other SMV isolates (Fig. 2B). The phylogenetic tree using PIPO protein sequences confirmed that SMV China is a member of SMV belonging to group A, which contains seven viruses including BPMV (Fig. 2C). Based on phylogenetic analyses, it seems that the consensus genome of SMV China is genetically close to the SMV isolates from South Korea.

Single nucleotide variations of SMV in the soybean seeds

It is well known that RNA viruses exhibit quasispecies nature, exhibiting several variants in the infected host. Therefore, we examined single nucleotide variations (SNVs) for SMV in the soybean seeds. The identified SMV China was used as a reference. After BWA alignment of raw data against SMV China, SNVs were identified using SAMtools (Fig. 3A). The SNVs in this study was derived from a population of different isolates. As a result, we identified 780 SNVs (Supplementary Table 1). SNVs were evenly distributed along the SMV genome (Fig. 3B). Most SNVs were Single nucleotide polymorphisms (SNPs) except one InDel (CAGG to CAGGAGG) at nt 640 of SMV China (Table S1). Four SNVs, C-U (190 SNVs), U-C (180 SNVs), A-G (168 SNVs), and G-A (155 SNVs), were frequently identified (Fig. 3C). Based on SNV results, the mutation rate for SMV in the soybean seeds was 8.2045%, indicating a high level of mutations for the SMV RNA genome. In addition, we calculated the ratio of Ts/Tv (Transition versus Transversion). The Ts/Tv ratio for SMV China was 8.06 (693/86).

The amount of viral RNA in the soybean transcriptome

It might be of interest to examine viral RNAs in the analyzed soybean transcriptome. Of 116,108 contigs, virus-associated contigs account for 0.068% (79 contigs). The length of total assembled contigs was 67,363,642 bp and the total length of virus-associated contigs 36,022 bp, accounting for 0.0535%. The amount of virus-associated reads accounts for 0.0529% (39,403/74,431,152) of reads. Moreover, we calculated SMV copy numbers within the soybean transcriptome resulting in 414 SMV virus copies, which is highly correlated with sequence coverage of SMV genome. This result indicates high variability of SMV genome.

Discussion

Development of NGS provides various DNA as well as RNA sequencing data (Metzker, 2010). The main purposes of DNA and RNA sequencing is elucidation of the genome and transcriptome of target eukaryotic and prokaryotic organisms (Morozova and Marra, 2008). In case of bacteria, metagenomics using 16s rRNA sequences that are highly conserved in bacteria species is intensively performed to study bacterial communities under specific conditions (Wang and Qian, 2009). However, viruses do not have any conserved sequences like bacteria, and genomes of viruses are mostly very small (Edwards and Rohwer, 2005). Therefore, virus-specific sequencing usually requires a purification step for NGS. For example, extraction of double-stranded RNAs from virus-infected organisms followed by NGS is one of the efficient approaches to identify viruses (Yanagisawa et al., 2016). Moreover, sequencing of small RNAs is an alternative technique for virus identification and genome assembly (Vodovar et al., 2011). In addition, RNA-Seq is also a good technique to identify viruses that have a poly(A) tail. However, several recent studies demonstrated that viruses and viroids without a poly(A) tail can be detected by RNA-Seq (Burger and Maree, 2015; Jo et al., 2016).
In this study, we identified several viruses infecting soybean. This transcriptome was initially conducted for expression profiling of soybean seed development. Thus, this transcriptome is not derived from a single condition but from six developmental seed stages in which several seeds might be included for total RNA extraction. Although we identified five viruses that might infect soybean, four viruses other than SMV were identified based on only one single contig, and their presence should be validated by other methods. In many cases, the partial viral sequence or contig is homologous to a closely related virus, not the target virus. Thus, it is possible that the identified virus-associated contigs might be not from the infected viruses but from other viruses which share similar viral sequences.
SMV is seed-borne and transmitted by aphids (Domier et al., 2011). Soybean seeds infected by SMV often display a discolored and mottled seed. In addition, BCMV is known as a seed-borne virus (Refugee et al., 1987). Seed-borne viruses can be actually infected in embryo, such as BCMV, or carried on the seed coat (Jafarpour et al., 1979). In addition, seed transmission of CMV has been identified in several plants such as pepper, spinach, and lupin (Ali and Kobayashi, 2010; Wylie et al., 1993; Yang et al., 1997). Based on previous knowledge on seed-borne viruses, the identification of SMV, BCMV, and CMV in the soybean seed is not surprising. In addition, the infection of LCV in green bean (Phaseolus vulgaris L.) has been recently reported (Ruiz et al., 2014). However, the infection of LIYV and LCV, which are members in the genus Crinivirus, in the soybean seed should be validated.
The soybean transcriptome was derived not from a single soybean seed but from a mixture of soybeans which were further divided into six developmental stages of seeds. The lengths of assembled contigs-associated with SMV in this study might be shorter than virus-associated contigs from a single plant due to the transcriptome containing several variants of SMV. Therefore, the assembled genome of SMV China is a consensus sequence of several SMV variants. Although the portion of SMV-associated sequences accounted for about 0.05% in the total transcriptome, the coverage of SMV genome in this study was about 414, and its coverage was also visualized by the alignment of raw data on the genome of SMV China. As a result, we could de novo assemble SMV genome based on enough sequence data associated with SMV.
Based on the assembled SMV genome, we could also identify SNVs for SMV. As we expected, we found several SNVs that resulted from a mixture of SMV infected diverse seed samples. However, we could not reveal the exact number of variants. Furthermore, the identification of SNVs in SMV demonstrated that not a specific region of SMV but several regions of SMV genome were highly mutated. The presence of several SMV variants in the soybean seeds is a very interesting finding, indicating that SMV is highly replicated in the developing seeds; this might be correlated with some disease symptoms in the soybean seeds caused by SMV. It might be of interest to examine replication rates of SMV in different developmental stages and tissues; this could provide evidence of the quasispecies nature of SMV in the near future.
Phylogenetic analyses suggested that the identified SMV isolate China was very different from other known SMV isolates based on polypeptide sequences. However, SMV isolate China seems to be highly correlated with two SMV isolates from South Korea, suggesting the phylogenetic correlation between geographical regions and SMV isolates.
Our SNV analysis in the soybean seeds indicates a high level of quasispecies nature for SMV. Mutations were not in a specific region but in most regions of SMV genome. Furthermore, we found that A-G and C-U conversions and vice and versa were frequent.
Taken together, our bioinformatics analyses using soybean seed transcriptomes identified five viruses infecting the soybean seeds. Of these five viruses, we de novo assembled the genome of SMV isolate China and analyzed SNVs revealing quasispecies nature of SMV in the soybean seeds for the first time. Our approaches and analyses in this study are valuable for the virus-associated studies using NGS-based transcriptome data.

Supplementary Information

Acknowledgments

This work was partially supported in part by the National Research Foundation of Korea (NRF) grant funded by the Korea government Ministry of Education (No. NRF-2016R1D1A1A02937216); the Agenda Program, the Rural Development Administration (RDA) (No. PJ01194803); and the Vegetable Breeding Research Center (No. 710001-05) through the Agriculture Research Center program from the Ministry for Food, Agriculture and Rural Affairs, Republic of Korea. WKC was supported by a research fellowship from the Brain Korea 21 Plus Project.

Fig. 1
De novo assembly of SMV isolate in China using transcriptome data. (A) Size distribution of virus-associated contigs. Red-colored bar indicates SMV-associated contigs. Four viruses with respective contig length were indicated. (B) Alignment of 79 SMV-associated contigs on the assembled genome of SMV isolate in China using BWA program. Black bar indicates the reference SMV genome. Sequence alignment was visualized by Tablet program. (C) Genome organization of SMV isolate in China. The nucleotide positions of two proteins, GP1 and GP2, were indicated.
ppj-33-478f1.gif
Fig. 2
Phylogenetic relationship of the assembled SMV isolate China with known SMV isolates. Phylogenetic trees of SMV isolates using complete genomes (A), polyproteins (B), and PIPO sequences (C). The respective genome and protein sequences were blasted against NCBI database and highly matched sequences were used for construction of phylogenetic trees using MEGA6 program using neighbor-joining method with 1000 bootstrap replications. Kimura 2-parameter and Poisson substitution model were used for nucleotide and protein sequences, respectively.
ppj-33-478f2.gif
Fig. 3
SNVs of SMV in the soybean seed transcriptome. (A) Raw data were mapped on the genome sequence of SMV isolate China using BWA and visualized by Tablet program. (B) The positions of identified single nucleotide variations on the SMV were visualized by Tablet program. Detailed information for SNVs can be found in Supplementary Table 1. (C) The numbers of identified SNVs of SMV in the soybean seed transcriptome.
ppj-33-478f3.gif
Table 1
Summary of de novo soybean transcriptome assembly using Trinity
Accession number  SRR1777405a
Total trinity transcripts 116108
Percent GC 43.97
Contig N50 710 bp
Median contig length 428 bp
Average contig 580.18 bp
 Total assembled bases  67363642 bp

a We assembled raw data from two different libraries using Trinity program.

The statistics of assembled contigs were calculated by TrinityStats.pl in the Trinity program.

Table 2
Summary of blast results to identify virus-associated contigs
Query id Subject id Name of virus Identity (%) Alignment length Mismatches Gap opens Query start Query end Subject start Subject end E value Bit score
TR2274|c0_g1_i1 NC_002634.1 Soybean mosaic virus 93.13 233 16 0 2 234 8571 8803 3.00E-93 342
TR3618|c0_g1_i1 NC_002634.1 Soybean mosaic virus 91.02 256 23 0 1 256 1342 1597 2.00E-94 346
TR3618|c0_g1_i2 NC_002634.1 Soybean mosaic virus 90.58 276 26 0 1 276 1342 1617 2.00E-100 366
TR3858|c0_g1_i1 NC_002634.1 Soybean mosaic virus 97.35 264 7 0 1 264 910 1173 2.00E-125 449
TR3858|c0_g1_i2 NC_002634.1 Soybean mosaic virus 96.6 235 8 0 1 235 939 1173 1.00E-107 390
TR4672|c0_g1_i1 NC_002634.1 Soybean mosaic virus 96.55 261 9 0 1 261 9036 9296 2.00E-120 433
TR4672|c0_g1_i2 NC_002634.1 Soybean mosaic virus 97.7 261 6 0 1 261 9036 9296 2.00E-125 449
TR5077|c1_g1_i1 NC_002634.1 Soybean mosaic virus 94.19 258 15 0 3 260 4680 4937 9.00E-109 394
TR5077|c1_g1_i2 NC_002634.1 Soybean mosaic virus 91.47 258 22 0 3 260 4680 4937 4.00E-97 355
TR5102|c0_g1_i1 NC_002634.1 Soybean mosaic virus 91.96 224 18 0 1 224 7552 7329 6.00E-85 315
TR5869|c0_g1_i1 NC_002634.1 Soybean mosaic virus 91.98 212 17 0 5 216 7243 7032 6.00E-80 298
TR5869|c0_g2_i1 NC_002634.1 Soybean mosaic virus 92.45 212 16 0 5 216 7243 7032 1.00E-81 303
TR5869|c0_g3_i1 NC_002634.1 Soybean mosaic virus 92.92 212 15 0 5 216 7243 7032 3.00E-83 309
TR5869|c0_g4_i1 NC_002634.1 Soybean mosaic virus 92.92 212 15 0 5 216 7243 7032 3.00E-83 309
TR7406|c0_g1_i1 NC_002634.1 Soybean mosaic virus 94.64 280 15 0 1 280 2677 2956 6.00E-121 435
TR7406|c0_g1_i2 NC_002634.1 Soybean mosaic virus 92.12 241 19 0 1 241 2677 2917 1.00E-92 340
TR7406|c0_g1_i3 NC_002634.1 Soybean mosaic virus 94.16 274 16 0 1 274 2677 2950 5.00E-116 418
TR7406|c0_g1_i4 NC_002634.1 Soybean mosaic virus 93.36 241 16 0 1 241 2677 2917 1.00E-97 357
TR8100|c0_g1_i1 NC_002634.1 Soybean mosaic virus 97.86 234 5 0 12 245 6060 6293 4.00E-112 405
TR9520|c0_g1_i1 NC_002634.1 Soybean mosaic virus 95.06 385 19 0 1 385 8268 7884 2.00E-172 606
TR9520|c0_g1_i2 NC_002634.1 Soybean mosaic virus 96.65 239 8 0 4 242 8122 7884 6.00E-110 398
TR9520|c0_g1_i3 NC_002634.1 Soybean mosaic virus 94.66 356 19 0 1 356 8268 7913 2.00E-156 553
TR9520|c0_g1_i4 NC_002634.1 Soybean mosaic virus 94.38 356 20 0 1 356 8268 7913 9.00E-155 547
TR9520|c0_g1_i5 NC_002634.1 Soybean mosaic virus 95.06 385 19 0 1 385 8268 7884 2.00E-172 606
TR9520|c0_g1_i6 NC_002634.1 Soybean mosaic virus 96.19 210 8 0 4 213 8122 7913 8.00E-94 344
TR9520|c0_g1_i7 NC_002634.1 Soybean mosaic virus 96.88 385 12 0 1 385 8268 7884 0 645
TR13605|c0_g1_i1 NC_002634.1 Soybean mosaic virus 92.25 400 31 0 10 409 8665 9064 8.00E-161 568
TR13605|c0_g1_i2 NC_002634.1 Soybean mosaic virus 94.75 400 21 0 10 409 8665 9064 2.00E-177 623
TR15892|c0_g1_i1 NC_002634.1 Soybean mosaic virus 92.64 231 17 0 2 232 5845 5615 2.00E-90 333
TR20496|c0_g1_i1 NC_002634.1 Soybean mosaic virus 96.88 224 7 0 1 224 2087 1864 3.00E-103 375
TR22770|c0_g1_i1 NC_002634.1 Soybean mosaic virus 91.67 240 20 0 1 240 6413 6652 2.00E-90 333
TR22770|c0_g1_i2 NC_002634.1 Soybean mosaic virus 92.53 281 21 0 2 282 6372 6652 2.00E-111 403
TR25078|c0_g1_i1 NC_002634.1 Soybean mosaic virus 88.54 253 29 0 1 253 8730 8478 1.00E-82 307
TR25078|c0_g2_i1 NC_002634.1 Soybean mosaic virus 94.72 246 13 0 16 261 8627 8382 2.00E-105 383
TR25078|c0_g2_i2 NC_002634.1 Soybean mosaic virus 93.7 349 22 0 1 349 8730 8382 2.00E-147 523
TR25078|c0_g2_i3 NC_002634.1 Soybean mosaic virus 95.72 187 8 0 43 229 8568 8382 5.00E-81 302
TR25078|c0_g2_i4 NC_002634.1 Soybean mosaic virus 90.91 253 23 0 1 253 8730 8478 1.00E-92 340
TR32819|c0_g1_i1 NC_002634.1 Soybean mosaic virus 91.7 265 22 0 2 266 2515 2251 6.00E-101 368
TR32819|c0_g2_i1 NC_002634.1 Soybean mosaic virus 92.08 265 21 0 2 266 2515 2251 1.00E-102 374
TR34507|c0_g1_i1 NC_002634.1 Soybean mosaic virus 87.27 377 44 4 4 378 3523 3149 1.00E-118 427
TR37651|c0_g1_i1 NC_002634.1 Soybean mosaic virus 87.61 218 24 3 2 218 410 625 2.00E-65 250
TR37651|c0_g3_i1 NC_002634.1 Soybean mosaic virus 87.27 487 57 4 2 487 410 892 1.00E-155 551
TR37706|c0_g2_i1 NC_002634.1 Soybean mosaic virus 90.51 274 24 2 1 273 1128 1400 9.00E-99 361
TR41793|c1_g1_i1 NC_002634.1 Soybean mosaic virus 92.89 394 28 0 1 394 7483 7876 2.00E-162 573
TR41793|c1_g1_i2 NC_002634.1 Soybean mosaic virus 93.15 438 29 1 1 437 7483 7920 0 641
TR41793|c1_g1_i3 NC_002634.1 Soybean mosaic virus 91.55 213 18 0 23 235 7486 7698 8.00E-79 294
TR41793|c1_g1_i4 NC_002634.1 Soybean mosaic virus 93.93 445 27 0 1 445 7483 7927 0 673
TR41793|c1_g1_i5 NC_002634.1 Soybean mosaic virus 91.59 226 19 0 1 226 7473 7698 2.00E-84 313
TR41793|c1_g1_i6 NC_002634.1 Soybean mosaic virus 93.03 445 31 0 1 445 7483 7927 0 651
TR41793|c1_g1_i7 NC_002634.1 Soybean mosaic virus 91.17 419 37 0 1 419 7473 7891 2.00E-161 569
TR44246|c0_g1_i2 NC_002634.1 Soybean mosaic virus 87.9 157 18 1 87 242 477 633 2.00E-45 183
TR44822|c4_g1_i1 NC_002634.1 Soybean mosaic virus 97.83 460 10 0 2 461 843 384 0 795
TR44822|c4_g1_i2 NC_002634.1 Soybean mosaic virus 97.65 765 18 0 2 766 843 79 0 1314
TR44822|c4_g1_i3 NC_002634.1 Soybean mosaic virus 97.27 622 14 1 2 623 843 225 0 1051
TR44822|c4_g2_i1 NC_002634.1 Soybean mosaic virus 90.13 1256 122 2 1 1255 1991 737 0 1631
TR44822|c4_g2_i2 NC_002634.1 Soybean mosaic virus 91.46 820 70 0 1 820 1918 1099 0 1127
TR44822|c4_g2_i3 NC_002634.1 Soybean mosaic virus 92.75 483 35 0 1 483 1991 1509 0 699
TR44822|c4_g2_i4 NC_002634.1 Soybean mosaic virus 88.67 256 29 0 1 256 1617 1362 2.00E-84 313
TR44822|c4_g2_i5 NC_002634.1 Soybean mosaic virus 93.9 246 15 0 19 264 1853 1608 4.00E-102 372
TR44822|c4_g2_i6 NC_002634.1 Soybean mosaic virus 94.81 231 12 0 19 249 1853 1623 9.00E-99 361
TR44822|c4_g2_i7 NC_002634.1 Soybean mosaic virus 94.15 410 24 0 1 410 1918 1509 5.00E-178 625
TR44822|c5_g1_i1 NC_002634.1 Soybean mosaic virus 95.98 994 40 0 2 995 5991 6984 0 1615
TR44822|c5_g1_i2 NC_002634.1 Soybean mosaic virus 94.11 3599 207 4 2 3596 5991 9588 0 5467
TR44822|c5_g2_i1 NC_002634.1 Soybean mosaic virus 93.3 224 15 0 4 227 8124 8347 6.00E-90 331
TR44822|c5_g1_i3 NC_002634.1 Soybean mosaic virus 96.21 501 19 0 2 502 5991 6491 0 821
TR44822|c5_g1_i4 NC_002634.1 Soybean mosaic virus 92.81 292 21 0 2 293 5991 6282 1.00E-117 424
TR44822|c6_g1_i1 NC_002634.1 Soybean mosaic virus 95.07 1015 50 0 1 1015 6049 5035 0 1598
TR44822|c6_g2_i1 NC_002634.1 Soybean mosaic virus 97.64 212 5 0 10 221 4930 4719 6.00E-100 364
TR44822|c6_g2_i2 NC_002634.1 Soybean mosaic virus 95.83 240 10 0 1 240 5051 4812 4.00E-107 388
TR44822|c6_g2_i3 NC_002634.1 Soybean mosaic virus 97.52 1372 34 0 1 1372 5146 3775 0 2346
TR44822|c6_g2_i4 NC_002634.1 Soybean mosaic virus 96.59 293 10 0 1 293 5146 4854 2.00E-136 486
TR44822|c6_g3_i1 NC_002634.1 Soybean mosaic virus 95.8 691 27 2 5 694 2746 2057 0 1114
TR44822|c6_g3_i2 NC_002634.1 Soybean mosaic virus 96.69 877 29 0 2 878 2822 1946 0 1459
TR44822|c6_g4_i1 NC_002634.1 Soybean mosaic virus 95.39 1149 53 0 1 1149 3889 2741 0 1829
TR44822|c6_g4_i2 NC_002634.1 Soybean mosaic virus 94.89 333 17 0 1 333 3598 3266 5.00E-147 521
TR45256|c0_g1_i1 NC_002634.1 Soybean mosaic virus 93.49 261 17 0 4 264 6897 6637 4.00E-107 388
TR45256|c0_g1_i2 NC_002634.1 Soybean mosaic virus 94.32 229 13 0 2 230 6865 6637 5.00E-96 351
TR47685|c0_g1_i1 NC_002634.1 Soybean mosaic virus 92.53 281 21 0 1 281 5082 5362 2.00E-111 403
TR47685|c0_g2_i1 NC_002634.1 Soybean mosaic virus 92.53 281 21 0 1 281 5082 5362 2.00E-111 403
TR44246|c0_g1_i1 NC_003397.1 Bean common mosaic virus 81.86 408 68 6 490 894 458 862 2.00E-91 339
TR19277|c0_g2_i1 NC_003617.1 Lettuce infectious yellows virus RNA1 75.34 146 29 6 467 607 6837 6694 1.00E-08 63.9
TR45572|c0_g2_i1 NC_012910.1 Lettuce chlorosis virus RNA2 87.96 191 22 1 15 205 8555 8366 1.00E-57 224
TR29303|c0_g1_i1 NC_002034.1 Cucumber mosaic virus RNA1 91.28 298 26 0 4 301 1334 1631 1.00E-112 407

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