Population genetic evolution analysis of tsaoko stripe mosaic virus and development of RT-PCR and real-time RT-PCR detection methods for strains discrimination

Tsaoko stripe mosaic virus (TkSMV) has been identified as the predominant viral pathogen of Amomum tsaoko in Yunnan province of China. This virus induced various foliar symptoms including chlorosis, stripe mosaic and necrosis, while some infected A. tsaoko plants remain asymptomatic. Comprehensive investigation​ of the population genetic structure and molecular detection protocols for TkSMV is crucial for elucidating its evolutionary dynamics, epidemiology, and disease control strategies. Phylogenetic analysis based on 15 full-length genomic sequences and 29 coat protein (CP) gene fragments revealed three distinct evolutionary lineages (S1, S2, S3) within the TkSMV population. Neutrality tests analysis indicates that the genetic polymorphism of the TkSMV population is consistent with a neutral evolution model (Tajima’s D > 0, not significant). The comparative evolutionary analysis demonstrated significant selective pressures across viral proteins: codon usage bias analysis revealed strong purifying selection (dN/dS < 1) in nine structural/functional proteins, eliminating deleterious mutations. The 9K gene exhibited the highest nucleotide diversity (π= 0.13567), suggesting its role in adaptive evolution. NIa-Pro protease showed the lowest diversity (π= 0.09497), indicating functional constraint. Remarkably, the CP gene displayed polarized evolutionary patterns. N-terminal region (aa 1-100) acted as a hypervariable hotspot, C-terminal region (aa 151-240) maintained evolutionary conservation. Furthermore, multiplex RT-PCR assays for accurate discrimination TkSMV-S1/S2/S3 strains in mixed infections were developed. These advancements provide technical foundations for​ epidemiological surveillance​ through strain-specific monitoring, seed certification programs​ for A. tsaoko cultivation.