Identification of two putative novel deltapartitiviruses and an enamovirus in coriander transcriptomes

Coriander is a herbaceous spice and condiment crop also known for its medicinal properties. The present study identified two putative novel deltapartitiviruses and an enamovirus tentatively named as Coriandrum sativum deltapartitivirus 1, 2 (CsDPV1, 2) and Coriandrum sativum enamovirus (CsEV) in the publicly available transcriptome-assembled contigs derived from coriander grown in India. CsDPV1 and 2 contained tripartite and bipartite genomes, respectively, with each genome segment encoding a single open reading frame (ORF). CsEV contained five ORFs encoding proteins P0, P1, P2, P3 and P5. Phylogenetic analysis revealed three distinct subgroups of deltapartitiviruses wherein CsDPV1 and 2 grouped in subgroup 3 and 1, respectively, whilst CsEV formed a distinct sub-clade within enamoviruses. Further, the presence of CsDPV2 in fruit samples of one of the cultivars from where the virus was identified was confirmed through RT-PCR assay and Sanger sequencing. The study highlights the need for further studies on understanding the importance and the biological properties of identified novel viruses.

Coriandrum sativum L., commonly known as coriander, is an economically important herbaceous spice crop belonging to the family Apiaceae.Though native to the Mediterranean and the Middle East, the crop is widely cultivated in different regions of the world.Leaves of coriander are generally used as flavouring agents in continental preparations like curries and soups whilst its fruits are used as condiments in pickle and curry powder preparation.Besides culinary uses, coriander fruits are valued for their medicinal properties in Ayurvedic medicine.Products like oleoresins and volatile oil derived from coriander fruits have huge demand in the global market (Sharma and Sharma 2012;Choudhary et al. 2019).India is amongst the leading producer and exporter of coriander in the world (Sharma and Sharma 2012).
In recent times, novel plant viral sequences are being increasingly discovered by probing the plant transcriptomeassembled contigs available in Transcriptome Shotgun Assembly (TSA) database of National Centre for Biotechnology Information (Bejerman et al. 2021(Bejerman et al. , 2022;;Bejerman and Debat 2022;Sidharthan et al. 2022aSidharthan et al. , 2022bSidharthan et al. , 2022c;;Sidharthan et al. 2023).In the present study, we mined the transcriptome-assembled contigs derived from leaves and fruits of three coriander cultivars (cvs.)'AgCr-1', 'CO-2' and 'Pant haritma' grown in India, and deposited in the NCBI under the Bioproject PRJNA472685 for putative novel viral sequences.In brief, the original coriander transcriptome libraries were prepared with the isolated total RNA from leaf and fruit samples of three coriander cultivars using Illumina TruSeq stranded mRNA sample Communicated by Ran Wang.
V. Kavi Sidharthan and Damini Diksha have contributed equally to the manuscript.preparation kit after poly-T-based mRNA enrichment.The libraries were, then, sequenced in Illumina's NextSeq500 platform to obtain paired-end reads.Raw reads were preprocessed using Trimmomatic v0.35 and de novo assembled using Trinity v2.5.1.The assembled contigs publicly available in NCBI were imported into Galaxy Australia server (Community 2022) and subjected to BLASTX search (evalue cutoff: 1e-5) against the reference viral proteins downloaded at https:// ftp.ncbi.nlm.nih.gov/ refseq/ relea se/ viral/.Only contigs longer than or equal to 500 nucleotides (nt) and sharing similarity with known plant viruses were regarded as plant viral contigs.The longest contig of an identified putative novel virus with length approximating the length of genome/genome segment of the identified virus group and containing intact open reading frame(s) (ORFs) across libraries was regarded as coding-complete genome/genome segment of that virus.ORF prediction in viral genomes, motif, transmembrane helix (TMH) prediction in and molecular weight estimation of viral genomeencoded proteins were performed as described in Sidharthan and Baranwal (2021) whilst -1 ribosomal frameshift site was predicted as described in (Sidharthan et al. 2022a).After MUSCLE alignment of viral protein sequences in MEGA7 v 7.0.26(Kumar et al. 2016), phylogenetic trees were constructed using maximum-likelihood (ML) method and the corresponding best-fit model with 1000 bootstrap replicates in IQ-TREE webserver (Trifinopoulos et al. 2016), and the resulting trees were visualised in MEGA7.Total RNA was isolated from fruits samples of coriander cvs.'CO-2' and 'Pant haritma' derived from the same lots that were originally used for transcriptome sequencing using Spectrum™ Plant Total RNA kit (Sigma, USA) following manufacturer's protocol.cDNAs were synthesised from the isolated 500 ng RNA using 2 μM of each random hexamers and oligo-dT primers using FIREScript RT cDNA synthesis kit (Solis Biodyne, Estonia) following manufacturer's protocol.To detect Coriandrum sativum deltapartitivirus 2 (CsDPV2), reverse transcriptase polymerase chain reactions (RT-PCR) were performed in 25 µL final reaction volume containing 50 ng of cDNA, 0.4 mM of each forward (5'-ATC CAC CGT TCA TCA CAA -3') and reverse (5'-TGC TCT GTA AGC CGA AAT C-3') primers (designed from the recovered CsDPV2 RNA1 sequence) and 1X Dream Taq PCR master mix polymerase (Thermo Scientific) with the following cycling conditions: initial denaturation at 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 58 °C for 45 s, 72 °C for 45 s followed by a final denaturation step at 72 °C for 10 s in a thermal cycler (Eppendorf Mastercycler nexus GX2 PCR system).PCR amplicons were visualised on 1.8% agarose gel, eluted, cloned and sequenced as described in (Diksha et al. 2023).
No motif was predicted in the encoded CP sequences when searched against Pfam database.Contigs corresponding to CsDPV1 RNA1, 2 and 3 identified across libraries shared 99.8 to 100%, 95.8 to 100% and 99.8% nt sequence identities, respectively, amongst each other (Fig. S1).
RT-PCR amplification of identified putative novel viral sequences in RNA isolated from fruit samples of cvs.'CO-2' and 'Pant haritma' obtained from the same lots that were used for transcriptome sequencing yielded a desired amplicon of 583 bp in 'CO-2' sample for CsDPV2 RNA1.Amplified sequence (accession number: OQ506517) shared 98.1% nt sequence identity with the recovered CsDPV2 RNA1 sequence.RT-PCR amplification of other identified novel viruses using at least two primers pairs for each virus did not yield positive bands even under a wide range of annealing temperatures.This might be because of the longterm storage of fruits or lesser proportion of infected fruits in the lot.Based on the consensus statement report by Simmonds et al. (2017), the identified putative novel viruses in the current study can be regarded as bona fide ones.As the viruses identified in the current study are solely based on transcriptome data analysis, except CsDPV2, host assignment should be regarded cautiously until further validation.Further studies on understanding the importance and biological properties of identified putative novel viruses are needed.

Fig. 1
Fig. 1 Genome organisation of Coriandrum sativum deltapartitivirus 1 and 2 (CsDPV 1 and 2) identified in the current study (a).Phylogenetic relationship of deltapartitiviruses identified in the current study with other partitiviruses based on RdRp (b) and CP amino acid

Fig. 2
Fig. 2 Genome organisation of Coriandrum sativum enamovirus (CsEV) identified in the current study (a).Phylogenetic relationship of CsEV with other enamo-and poleroviruses based on P3 amino