High-Throughput Sequencing Identified Multiple Fig Viruses and Viroids Associated with Fig Mosaic Disease in Iraq

Abstract

Mosaic is the most common viral disease affecting fig plants. Although the Fig mosaic virus is the leading cause of mosaic disease, other viruses are also involved. High-throughput sequencing was used to assess viral infections in fig plants with mosaic. The genomic DNA and total RNAseq of mosaic-symptomatic fig leaves were sequenced using the Illumina platform. The analysis revealed the presence of fig badnavirus 1 (FBV-1), grapevine badnavirus 1 (GBV-1), citrus exocortis viroid (CEVd), and apple dimple fruit viroid (ADFVd). The FBV-1 and GBV-1 sequences were 7,140 bp and 7,239 bp long, respectively. The two genomes encode one open reading frame containing five major protein domains. The viroids, CEVd and ADFVd, were 397 bp and 305 bp long. Phylogenetic analyses revealed a close relationship between FBV-1 and Iranian isolates of the same species, while GBV-1 was closely related to Russian grapevine badnavirus isolates (Tem64, Blu17, KDH48, and Pal9). CEVd was closely related to other Iraqi isolates, while ADFVd was strongly related to a Spanish isolate. A registered endogenous pararetrovirus, caulimovirus-Fca1, with a size of 7,556 bp, was found in the RNA transcripts with a low expression level. This integrant was also detected in the genomes of the two lines 'Horaishi' (a female line) and 'Caprifig 6085' (a male line). Phylogenetic analyses revealed that caulimovirus-Fca1 was distinct from two other clades of different endogenous virus genera.

Keywords: apple dimple fruit viroid; citrus exocortis viroid; fig badnavirus 1; grapevine badnavirus 1; high-throughput sequencing.

Fig. 1

Severe mosaic symptoms on fig leaves caused by Fig mosaic virus in mixture with other viruses.

Fig. 2

Mapping DNA and RNA reads over the entire sequence of fig badnavirus 1 isolate Hilla showed full coverage and high assembled reads in DNA, while the RNA reads counts were fewer than DNA but covered the specific regions of coding sequence. The virus sequence encodes five major protein domains (DUF1319, Smc, RT_LTR, RNase_HI_RT_Ty3, and RT_RNaseH_2).

Fig. 3

Mapping DNA and RNA reads over the entire sequence of grapevine badnavirus 1 isolate Iraq showed full coverage and high assembled reads in DNA, and little reads in RNA. The virus sequence encodes five major protein domains (DUF1319, SbcC, RT_LTR, RVT_1RNase_HI_RT_Ty3, and ZnF_C2HC).

Fig. 4

(A) Fig badnavirus 1 maximum likelihood with 1,000 bootstrap replicates reconstructed based on 15 related virus genomes shows close relationship between the Iraqi isolates Hilla and Iraq. The outgroup member was Tobacco mosaic virus. (B) Fig badnavirus 2 maximum likelihood with 1,000 bootstrap replicates reconstructed based on 15 related virus genomes, the virus positioned within cluster of grapevine badna F1 isolates, Tobacco mosaic virus was the outgroup member.

Fig. 5

(A) Citrus exocortis viroid (CEVd) maximum likelihood with 1,000 bootstrap replicates tree reconstructed based on complete 18 CEVd isolates showed high similarity between the Iraqi isolates especially Najaf and Iraq. (B) The maximum likelihood with 1,000 bootstrap replicates tree of Apple dimple fruit viroid that reconstructed based on complete genome sequences of 15 related viroid isolates obtained from NCBI-GenBank, and citrus exocortis viroid was the outgroup member.

Fig. 6

The assembled reads of DNA to caulimovirus-FCa1 that encoded five protein domains and poorly found in RNA transcripts. The sequence of endogenous pararetrovirus (EPRV) encodes five protein domains: RNase-H, RT, RT-LTR, RVT-1, and MP.

Fig. 7

The maximum likelihood with 1,000 bootstrap replicates tree of caulimovirus-FCa1 that reconstructed based on complete genome sequences of 24 related integrants and Squash vein yellowing virus was the outgroup member.

Fig. 8

The abundance of each virus and viroids analysed in the study based on the assembled reads of each. Assembled reads showed nearly equal numbers of badnaviruses in DNA and RNA, but there was a higher proportion of fig badnavirus 1 (FBV-1) in RNA. Citrus exocortis viroid (CEVd) existed more than apple dimple fruit viroid (ADFVd), whereas Caulimovirus-FC1 was abundant in DNA but almost absent in RNA.