Abstract
Iranian johnsongrass mosaic virus (IJMV, genus Potyvirus, family Potyviridae) is one of the most prevalent viruses causing maize mosaic disease in Iran. In this study, the complete genomes (9,618 and 9,543 nucleotides) of two highly divergent IJMV isolates (Maz2 and Maz3) were obtained from the metagenomic analysis of Zea mays RNAs using Illumina sequencing. The genome contained a single open reading frame (9,165 nucleotides) encoding a polyprotein of 3,054 amino acids, flanked by a 5′-untranslated region (UTR) of 216 and 143 nucleotides and a 3′-UTR of 237 and 235 nucleotides. A comparative analysis of the complete genome showed that IJMV-Maz2 and Maz3 had 85.99% nucleotide and 94.56% amino acid sequence identity with each other and shared 84.87–88.74% nt and 94.24–96.17% aa identity with those of two other IJMV isolates available in the GenBank. The coat protein of Maz2 and Maz3 showed 86.40–95.72% nt sequence identity (90.79–97.70% aa identity) to 12 other IJMV isolates available in GenBank. Our results indicated a relatively stable and conserved genomic composition with a low codon usage bias in all of the assayed IJMV coding sequences. Analysis of various population genetics parameters and distribution of synonymous and nonsynonymous mutations revealed that purifying selection pressure was the major force acting upon the IJMV genome. The outcome of the study provides valuable insights on the evolution of IJMV genome, for which there are few genome sequences available, and informs the current breeding efforts towards resistance for IJMV.
Keywords: RNA-seq, IJMV, Complete genome, Phylogenetic analysis, Natural selection
Potyviruses are one of the major limitations in cereal production in Iran, as they account for severe yield reduction within smallholder farms. Iranian johnsongrass mosaic virus (IJMV) is a member of the genus Potyvirus (family Potyviridae), causes large economic losses of maize in Iran [17–19]. Like other potyviruses, it has a flexuous filamentous particle of 750 nm, and a single-stranded positive-sense RNA genome of about 9.6 kb. The viral genome is translated into a large polyprotein consisting of 3,054 amino acids which cleaved into ten multifunctional proteins [19, 24]. An additional peptide, P3N-PIPO, is translated from an overlapping ORF after + 2 frameshifting of the P3 cistron [6]. The occurrence of IJMV was first reported in johnsongrass in 1983 and then it has spread to the maize and sugarcane-growing regions of Iran. It can be transmitted by mechanical inoculation, as well as by several aphid species in a non-persistent manner. IJMV is listed as a SCMV subgroup and has a narrow host range limited to the Gramineae [19]. Understanding the evolution of viruses and the distribution of genetic diversity among individuals, populations, and gene pools is essential for efficient management and plant breeding programs [9]. As there is a direct correlation between virus evolution and durability of the resistance within the different cultivars, therefore, this information provides a good indicator of the possible duration of resistant cultivar being developed. In this study, the RNA deep-sequencing technology was employed for virus identification. In June 2019, leaf samples of maize plants showing virus-like disease symptoms (such as severe mosaic and dwarf) were collected from Mazandaran province, north of Iran (Fig. S1). To determine the causal agent(s), total RNA was extracted from the symptomatic leaf samples using SV Total RNA Isolation Kit (Promega, USA) on a pooled sample collected from seven diseased maize plants. Complementary DNA libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo-Zero rRNA Removal kit (Plant Leaf) following manufacturer’s instructions and sequenced using an Illumina NovaSeq 6000 (Macrogen, Seoul, South Korea). To identify possible viruses, RNA Seq data were trimmed, and low-quality bases and adaptor sequences were removed. Clean reads of symptomatic plants were mapped to reads from healthy-looking plant and reference genome (www.plants.ensembl.org) by CLC Genomics Workbench 20 (CLC Bio, Qiagen). Unmapped reads were saved and used for the genome De Novo assembly. Assembled contigs were analyzed using BLASTn searches by Geneious Prime v. 2019.1.3 (Biomatters Ltd., Auckland, New Zealand) to identify sequence similarities to reference viral genomes in the GenBank database. Two long contigs (10,437 bp and 9,600 bp in length) were identified from the assembled non-plant reads that mapped to IJMV sequences in the GenBank. These contigs were used to interrogate NCBI databases by BLASTn and BLASTx to identify sequences with shared identity to known viruses. Open reading frames (ORF), mature peptides, and domains encoded by them were predicted within Geneious Prime v. 2019.1.3 (Biomatters), the NCBI Conserved Domain Database (CDD), and InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan). ClustalW multiple sequence alignments and comparison of nucleotide and amino acid sequence identities were carried out using the Geneious Prime. The aligned sequences were assessed for putative recombination events using the different programs in the RDP4 package [16]. The phylogenetic analysis of the IJMV sequences was considered using the neighbor-joining (NJ) method in MEGAX [14]. Branch support was evaluated by Kimura’s two-parameter option [12], which was used to calculate 1000 bootstrap replications for NJ analysis. All positions containing gaps and missing data were eliminated. The nucleotide diversity (π) value, nonsynonymous substitution rate (dN), synonymous substitution rate (dS), and synonymous codon usage bias (CUB) were calculated using the DnaSP6 [22]. The ENC (effective number of codons) values were used to infer the magnitude of CUB in the IJMV coding regions and range from 20 (if only one codon is used for each amino acid, i.e., the codon bias is maximum) to 61 (if all synonymous codons for each amino acid are equally used, i.e., no codon bias) [23]. The fixed-effects likelihood (FEL), single-likelihood ancestor counting (SLAC), mixed effects model of evolution (MEME) (with significance levels set at P = 0.1), and random-effects likelihood (REL) (with Bayes factor = 50) methods as implemented in Datamonkey (www.datamonkey.org) [13], were used to determine the site-specific selection pressures acting on each codon of IJMV genome. Blastn search identified two single contigs of about 10 kb from the assembled non-plant reads that mapped to IJMV sequences in the GenBank. In the statistical mapping analysis of RNA-seq data, 41,506,790 151-nt raw reads were obtained for a total of 6,267,525,290 bases in infected maize plant. A total of 10,026,330 IJMV sequence reads mapped to the final contig obtained from maize. To confirm the paired-end RNA sequencing result of the contig sequences and to find the sample infected with IJMV, reverse-transcription PCR and Sanger sequencing were done with IJMV-specific primers [19]. Sequences obtained by the Sanger method were > 99% identical to those assembled from Illumina data, confirming that the sequences of the virus were correct in the regions tested. The two largest of these contigs were used to interrogate NCBI databases by BLASTn and BLASTx to identify sequences with shared identity to known viruses.
The verified complete genome for the IJMV-Maz2 (GenBank accession no. MN535992) and -Maz3 (MN535993) comprised 9,619 and 9,544 nucleotides respectively, excluding the 3′-terminal poly(A) tail. The new IJMV sequences harbor all the hallmarks of a typical member of the genus Potyvirus [1]. The ORF encoded a single large polyprotein, which is proteolytically processed into 10 functional proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, CP, and a truncated frameshift product, the PIPO protein. The ten putative mature proteins of two new isolates contained the conserved motifs with known functions for other potyviruses as described previously [19]. Among four fully sequenced IJMV isolates, only Maz3 had DAG motif rather than DVG motif.
The identities between the complete genomes of isolates Maz2 and Maz3 were 85.99% at nucleotide (nt) level and 94.56% at amino acid (aa) level. Maz2 and Maz3 shared nt identity of 84.87–88.74% (94.24–96.17% aa identity) with those of two other fully sequenced IJMV isolates, the highest with the isolate Maz-Bah (KT899778) (Table 1). Comparison of the ORF sequences between the IJMV isolates revealed values ranging from 84.71 to 88.65% nt identity. Maz2 shared nt identities of 78.97–97.57% with two other IJMV isolates for individual genes in the polyprotein, and Maz3 shared nt identities of 78.40–97.15%. The P1, 6K1, NIa-Vpg, NIa-Pro and CP of Maz2 were more identical to the Shz isolate (from johnsongrass), while the remaining six proteins and 5′-, 3′-UTRs showed higher identities to the Maz-Bah isolate (Table 1). The P1 of Maz3 was more identical to the Shz isolate, while the remaining ten proteins and 5′-, 3′-UTRs showed higher identities to the Maz-Bah isolate (Table 1).
Table 1.
Percent nucleotide/amino acid sequence identity of the complete genome, polyprotein and individual coding and non-coding region of IJMV-Maz2, and other IJMV isolates
| Isolate | 5′-UTR | P1 | HC-Pro | PIPO | P3 | 6k1 | CI | 6K2 | NIa-VPg | NIa-Pro | NIb | CP | 3′-UTR | Polyprotein | Whole genome |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Maz3 | 93.01/- | 82.12/81.97 | 90.72/96.96 | 97.15/96.34 | 89.34/97.41 | 83.08/94.03 | 83.75/96.87 | 82.39/94.34 | 84.13/95.24 | 84.16/95.45 | 83.88/94.24 | 87.28/92.11 | 92.86/- | 85.70/ 94.56 | 85.99 |
| (100/-) a | (100/100) | (100/100) | (100/100) | (100/100) | (100/100) | (100/100) | (100/100) | (100/100) | (100/100) | (100/100) | (100/100) | (100/-) | (100/100) | (100) | |
| Maz-Bah (KT899778) | 84.62/- | 78.97/81.12 | 89.28/97.17 | 97.57/96.34 | 89.82/97.69 | 81.59/97.01 | 93.73/97.81 | 83.65/92.45 | 82.89/95.24 | 81.96/94.63 | 85.99/94.43 | 87.83/92.43 | 92.86/- | 87.54/ | 87.63 |
| (81.12/-) | (78.40/79.83) | (89.13/97.17) | (97.15/95.12) | (93.66/98.56) | (91.54/97.01) | (85.27/97.49) | (88.05/98.11) | (91.71/98.94) | (89.39/98.76) | (87.72/95.97) | (95.72/97.70) | (96.61/-) | 94.79(88.65/96.17) | (88.74) | |
| Shz (JQ692088) | 83.22/- | 81.26/80.69 | 85.87/95.00 | 95.12/89.02 | 87.32/95.68 | 83.08/92.54 | 85.01/96.71 | 83.65/92.45 | 83.07/97.35 | 83.88/96.69 | 84.26/95.20 | 88.49/93.75 | 91.60/- | 85.06/94.43 | 85.20 |
| (82.52/-) | (80.11/81.55) | (86.09/95.00) | (94.72/87.80) | (85.59/95.39) | (81.59/97.01) | (85.58/97.02) | (81.13/94.34) | (85.01/94.71) | (80.17/95.87) | (84.13/94.05) | (89.04/93.75) | (92.37/-) | (84.71/94.24) | (84.87) |
aNumber outside the parenthesis showed the identity of Maz2 to other IJMV isolates, number inside the parenthesis showed the identity between Maz3 and other IJMV isolates
In recent studies, we confirmed that as the identities of the CP nt and aa sequences between IJMV and Zea mosaic virus (ZeMV) are more than the threshold of species demarcation, so probably both are the strains of the same virus. Accordingly, in this study ZeMV isolate was considered along with IJMV isolates. Isolates Maz2 and Maz3 also shared 86.40–95.72% nt identity (90.79–97.70% aa identity) in the CP coding region with 12 other IJMV isolates which CP sequences of them are available (see Table 2).
Table 2.
Pairwise sequence comparisons depicting nucleotide and amino acid sequence identities among CP coding region of IJMV isolates plus ZeMV
| Isolates | Maz2 | Maz3 | ||
|---|---|---|---|---|
| nt identity (%) | aa identity (%) | nt identity (%) | aa identity (%) | |
| Maz2 | 100 | 100 | 87.28 | 92.11 |
| Maz3 | 87.28 | 92.11 | 100 | 100 |
| Maz-Bah (KT899778) | 87.83 | 92.43 | 95.72 | 97.70 |
| Maz-B3 (KU746863) | 86.84 | 90.79 | 92.11 | 95.72 |
| GSr (KU746861) | 88.05 | 92.11 | 88.38 | 92.76 |
| GJ (KU746860) | 88.05 | 91.45 | 88.27 | 91.78 |
| KhuzJ (KU746862) | 88.93 | 93.09 | 89.04 | 93.75 |
| SCRA1-6 (KU746864) | 90.13 | 94.74 | 88.38 | 92.76 |
| SCRA1-7 (KU746865) | 90.57 | 92.11 | 91.45 | 96.38 |
| SCRA1-910 (KU746868) | 90.79 | 92.76 | 91.67 | 97.04 |
| SCRA1-13 (KU746866) | 91.89 | 95.07 | 88.27 | 92.43 |
| SCRA1-15 (KU746867) | 90.79 | 92.43 | 91.67 | 96.71 |
| Shz (JQ692088) | 88.49 | 93.75 | 89.04 | 93.75 |
| ZeMV (AF228693) | 86.95 | 91.78 | 86.40 | 91.12 |
The π value for the entire polyprotein sequences of four IJMV isolates was 0.15442, indicating high genetic diversity. Among all the coding regions analyzed here, the P1 possessed the greatest nucleotide diversity (π = 0.23093). This finding is in line with that of other studies [2, 20], showing that the P1 is the most variable protein among potyviruses. In recombination analysis using RDP4, no significant recombination event was detected in the sequences of IJMV isolates (data not shown).
The dN/dS (ω) ratio for the complete ORFs was less than 1.0 (dN/dS = 0.0275), indicating that the IJMV genome is under dominant purifying selection. As depicted in Table 3, the dN/dS ratio (ω) for each cistron, calculated separately, ranged from 0.014155 to 0.610975, suggesting the purifying selective pressure was not evenly distributed across the IJMV coding regions and distinct constraints affect different parts of the genomes. The strongest purifying selection was observed in the CI protein, supported by the smallest ω value (0.014155), while the weakest purifying selection was in the PIPO followed by the P1 protein, with the largest ω values (0.610975 and 0.09856, respectively).
Table 3.
Analysis of codon usage bias, genetic polymorphism and selection parameters acting on different coding regions of four IJMV isolates
| Genomic region | Size in nucleotide | ENC | CBI | π | dN | dS | dN/dS |
|---|---|---|---|---|---|---|---|
| P1 | 699 | 54.502 | 0.311 | 0.23093 | 0.10615 | 1.07699 | 0.09856 |
| HC-Pro | 1380 | 49.479 | 0.331 | 0.13350 | 0.01700 | 0.86873 | 0.019568 |
| P3 | 1041 | 51.540 | 0.342 | 0.12200 | 0.01565 | 0.68971 | 0.022690 |
| PIPO | 243 | 59.776 | 0.456 | 0.03975 | 0.03496 | 0.05722 | 0.610975 |
| 6K1 | 201 | 42.983 | 0.555 | 0.18446 | 0.02411 | 0.68707 | 0.035091 |
| CI | 1914 | 48.623 | 0.336 | 0.15068 | 0.01469 | 1.03779 | 0.014155 |
| 6K2 | 159 | 44.950 | 0.590 | 0.18768 | 0.02831 | 0.64423 | 0.043943 |
| NIa-VPg | 567 | 49.461 | 0.381 | 0.16668 | 0.01859 | 1.29813 | 0.014320 |
| NIa-Pro | 726 | 52.115 | 0.354 | 0.18645 | 0.01806 | 0.67046 | 0.026936 |
| NIb | 1563 | 48.782 | 0.309 | 0.16760 | 0.02661 | 1.16967 | 0.022750 |
| CP | 915 | 52.033 | 0.329 | 0.11456 | 0.02907 | 0.55068 | 0.052789 |
| aCP (IJMV + ZeMV) | 915 | 52.671 | 0.324 | 0.12662 | 0.03359 | 0.61765 | 0.054383 |
aAnalysis of CP gene of IJMV isolates plus ZeMV
The divergence value of IJMV polyprotein nt sequences was 0.12662 which is reduced by purifying (negative) selection pressure, as has been reported for other potyviruses [20, 21]. Different selective constraints imposed on different IJMV proteins might be correlated with the various functions of these proteins in the infection cycle of the virus and/or their interactions with the host and aphid vectors [8, 20].
Our findings also revealed most of the codons were under negative selection or neutral evolution, however, a few codons in IJMV HC-Pro (site 160), P3 (site 319), CI (sites 474 and 551), NIa-VPg (site 172), and CP (sites 44 and 51) were found under positive selection only by MEME method. The presence of episodic positive or diversifying selection in some coding regions reflects the molecular adaptation to the plant or the aphid vectors [15, 20].
The ENC values among the present IJMV isolates were high and ranged from 42.983 to 59.776 (Table 3). The higher ENC values in IJMV coding regions indicated low CUB, resulting in higher genomic stability in these genes. Compared with separate coding sequences, the lower ENC values for the 6K1 gene suggested a slightly greater codon bias than was observed for the other genes.
The low CUB might be beneficial to IJMV on its fitness to the host species with potentially distinct codon preferences. Low CUB was also observed in several RNA viruses, such as Citrus tristeza virus [4], Hepatitis C virus [10], Ebola virus [7], and Zika virus [5]. A low CUB of RNA viruses has an advantage for efficient replication in the host cells by reducing the competition between the virus and host in using the synthesis machinery [11].
To determine the codon usage bias of IJMV genes, the codon bias index (CBI) for every protein-coding gene in the genome was also calculated. CBI ranges from − 1, indicating that all codons within a gene are nonpreferred, to + 1, indicating that all codons are the most preferred, with a value of 0 indicative of random use [3]. As shown in Table 3, all IJMV genes have positive CBI values with an average CBI of 0.39, indicating a strong preference for optimal codons. Phylogenetic tree analyses based on the multiple alignments of the entire nt and aa sequences suggested two clades. Phylogroup I consisted of three IJMV isolates from maize and phylogroup II included johnsongrass isolate of IJMV. Phylogenetic analyses based on complete nucleotide sequences of the CP coding region classified all IJMV isolates into two groups and four subgroups. Group I included maize and sugarcane isolates of IJMV. Group II included IJMV isolates from sorghum and johnsongrass plus ZeMV isolate from Israel (Fig. 1). A phylogenetic tree based on the CP aa sequences of the same isolates was slightly different in which Maz2, SCRA1-13, and SCRA1-6 were clustered with isolates in group II (Fig. 2). Here, we determined the complete genome sequences and molecular genetic characteristics of two IJMV isolates from maize, assembled by Illumina deep RNA sequenced reads and RT-PCR. Our results provide more evidence that the genome of IJMV is highly variable. The genetic variability of maize-infecting isolates of IJMV reported in this study may provide a foundation for developing effective control measures to prevent severe losses and make amendments in management strategies of different crops. The biological implications of this sequence divergence remain to be elucidated.
Fig. 1.
Neighbor-joining phylogenetic trees based on alignments of nucleotide (up) and amino acid (down) sequences of complete genome of four IJMV isolates. A sorghum mosaic virus (SrMV) isolate was used as outgroup. Isolates are indicated in the tree by accession number/isolate name/host/origin. Numbers at each node indicate bootstrap percentages based on 1000 replications. The marked isolates were obtained in this research. Evolutionary analyses were computed using the Kimura2 parameter method in MEGA X
Fig. 2.
Phylogenetic relationships of coat protein nt (left) and aa (right) sequences of IJMV isolates plus ZeMV. The CP sequence of sorghum mosaic virus isolate XoS was used as an outgroup. Numbers at each node indicate bootstrap percentages based on 1000 replications
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Funding
This study was financially supported by grant number 49211 from Ferdowsi University of Mashhad.
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Conflict of interest
The authors declare that they have no conflict of interest.
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Supplementary Information
The online version contains supplementary material available at(10.1007/s13337-021-00671-w).
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