Genome-wide identification of rhabdoviral sequences in alfalfa (Medicago sativa L.)

Authors: Grinstead, Samuel; Nemchinov, Lev

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 5/22/2026
Publication Date: 5/22/2026
Citation: Grinstead, S.C., Nemchinov, L.G. 2026. Genome-wide identification of rhabdoviral sequences in alfalfa (Medicago sativa L.) . bioRxiv. Article e726541. https://doi.org/10.64898/2026.05.20.726541.
DOI: https://doi.org/10.64898/2026.05.20.726541

Interpretive Summary: Integrated virus elements (EVEs) are viral genetic sequences that have become permanently embedded in the host plant’s genome over evolutionary time. In crops, these genetic fossils provide important evolutionary context, may act as natural defense mechanisms against exogenous viruses, and serve as valuable tools for crop improvement and biotechnology. Our previous discoveries of pervasive sequences belonging to agriculturally important rhabdoviruses in both healthy and infected alfalfa plants prompted us to suggest that the alfalfa genome might be populated with integrated rhabdoviral sequences. Bioinformatics analysis confirmed the hypothesis, detecting multiple rhabdoviral sequences across 26 alfalfa genomes. It is expected that this study will be of interest to scientists involved in research on alfalfa improvement, plant pathologists, and virologists.

Technical Abstract: Endogenous viral elements (EVEs) are viral sequences integrated into host chromosomes and inherited by the host progeny as genetic alleles. Plant genomes contain two main types of EVEs: endogenous pararetroviruses (EPRVs), which originate from reverse-transcribing dsDNA viruses (Caulimoviridae), and endogenous non-retroviral elements (ENREs), which are derived from ssDNA, ssRNA, and dsRNA viruses. While integration of endogenous pararetroviruses into plant genomes is characterized relatively well, insertions of non-retroviral elements (ENREs) remain largely obscure. It is possible that pararetroviruses or retrotransposons provide the reverse transcriptase required to facilitate this integration. As of today, no ENREs were reported in alfalfa (Medicago sativa L.). In this work, we performed bioinformatics analysis to identify the potential presence of endogenous rhabdoviral elements in 26 publicly available alfalfa genomes. The analysis identified sequences of rhabdoviral nucleocapsid protein (N) ranging in size from 1065-1069 in chromosome 3 of five genomes; 1016-1022 nt in Chr 4 of 8 genomes; and from 1078-1388 nt in Chr 5 of all 26 genomes. Individual analysis of all detected EVEs via BLASTX searches against the ClusteredNR database identified sequences that shared 89–100% identity (E-value=0) and 89-100% query coverage with the nucleocapsid proteins of two previously characterized viruses: alfalfa nucleorhabdovirus 1 (ANRV1, UGO47147.1) and alfalfa cytorhabdovirus 1 (ACRV1, UGO47148.1). Phylogenetic analysis performed with MEGA 12 software clustered ANRV1 and ACRV1 EVEs with representative members of the genera Betanucleorhabdovirus and Betacytorhabdovirus, respectively. As both viruses are phylogenetically distinct from other representatives of the family, this may suggest that their EVEs are uniquely adapted to Medicago sativa. Consequently, they may have evolved and potentially acquire functional roles in alfalfa’s development and response to environmental stresses. We believe this study reveals the first documented case of rhabdoviruses integrated into the genomes of M. sativa. Furthermore, this research indicates that rhabdoviruses previously identified in alfalfa could represent endogenous viral elements integrated into the plant genome.