Complete genome sequence analysis of grapevine pinot gris virus isolates from Inner Mongolia

Abstract:【Objective】The objective of this study was to acquire the complete genomic sequence of grapevine pinot gris virus (GPGV) isolates from Inner Mongolia and to perform a comprehensive analysis of the GPGV population, encompassing sequence identity, phylogenetic relationships, gene recombination, sequence similarity and genetic diversity.【Methods】Grape leaf samples that had previously tested positive for GPGV served as the experimental materials for total RNA extraction. A total of 100 mg of GPGV-infected grape samples were processed in accordance with the Spectrum™ Plant Total RNA Kit instructions. The quality and concentration of the extracted RNA were evaluated via 1% agarose gel electrophoresis and microspectrophotometry, respectively, and the RNA was preserved at -80 ℃ for future use. Vector NTI software was utilized to align all the full-length genomic sequences of GPGV reported in the NCBI GenBank database. Three primer pairs (GPGV-1F/GPGV-1R, GPGV-2F/GPGV-2R and GPGV-3F/GPGV-3R) were designed within the conserved regions to amplify the complete genomic sequence of GPGV, ensuring that overlapping fragments between adjacent amplification products exceeded 200 bp. Subsequently, primers (GPGV3 and GPGV1) were designed for amplifying the terminal sequences of GPGV. Total RNA was employed as a template to synthesize cDNA using the SuperScript™ Ⅲ Reverse Transcriptase Kit under the conditions of 50 ℃ for 1 hour, followed by 70 ℃ for 15minutes. The cDNA template was then used to amplify the nucleotide sequences of GPGV with Q5 High-Fidelity 2×Master Mix, employing thermal cycling parameters of denaturation at 98 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, and extension at 72 ℃ for 2 minutes over 35 cycles. The SMARTer® RACE 5'/3' Kit was used to amplify the 5' and 3' terminal sequences of GPGV. PCR products were identified through 1% agarose gel electrophoresis, and the target fragments were purified using a gel DNA purification kit. The amplified and RACE-obtained GPGV genomic sequences were assembled using Vector NTI software to reconstruct the complete genomic sequences of GPGV. ClustalW in MEGA 11 was employed to conduct multiple sequence alignments of all complete genomic sequences of GPGV in the NCBI database (152 isolates), and a phylogenetic tree was constructed using the Maximum-Likelihood method with 1000 bootstrap replicates as determined by the MODLES program. Sequence identity analysis was performed on the complete genomic sequences and open reading frames (ORFs) using BioEdit 7.2 software. Recombination analysis was executed on the complete genomic sequences of the isolates using seven recombination detection algorithms provided by RDP4 software. Population neutrality tests, selection pressure analysis, nucleotide polymorphism analysis, and haplotype polymorphism analysis were conducted on GPGV isolates using DnaSP v.6.12.03.【Results】The complete genomes of two GPGV isolates from Inner Mongolia (20IM- ViVi1 and 20IM- ViVi2) were successfully cloned, each comprising 7250 nucleotides and encoding three open reading frames (ORFs). Sequence identity analysis demonstrated that the genome sequences of isolates 20IM-ViVi1 and 20IMViVi2 were 96.4% identical. Their identity with other isolates varied from 79.7% to 96.8% and 79.5% to 97.7%, respectively. Furthermore, the identity among the full genome sequences of GPGV isolates in China spanned from 82.0% to 99.9%. A phylogenetic tree based on the complete genome sequences of all GPGV isolates revealed that the existing 152 complete genomes were partitioned into four clades. Clade Ⅲ and Clade Ⅳ were further divided into two minor subclades (a and b). The two isolates 20IMViVi1 and 20IM-ViVi2 obtained in this study were both grouped in CladeⅠ and were most closely related to the Summer Black grape isolate SRR2845691-GPGV from China. A phylogenetic tree constructed from Chinese GPGV isolates indicated that these isolates were predominantly divided into four clades. The GPGV isolate Shihezi-1 from Xinjiang displayed a relatively high genetic distance from other isolates, with a distant phylogenetic relationship, and was therefore placed in CladeⅠ separately. The isolates 20IM-ViVi1 and 20IM-ViVi2 obtained in this study were both aggregated in Clade Ⅳ and were most closely related to the Summer Black grape isolate SRR2845691-GPGV. Recombination analysis revealed that no significant recombination events were detected in the GPGV isolates 20IM-ViVi1 and 20IM-ViVi2. Genetic diversity analysis suggested that GPGV possessed high genetic diversity, with the Asian isolates showing the highest genetic diversity.【Conclusion】This study marks the first to obtain the complete genome sequences of GPGV isolates from Inner Mongolia. Both GPGV isolates had a genome length of 7250 nt, containing three ORFs, and exhibited high identity with existing GPGV isolates (excluding the Japanese isolate H-JP2), with identity ranges from 79.7% to 96.8% and 79.5% to 97.7%, respectively. Additionally, the phylogenetic tree constructed from all GPGV complete genome sequences was divisible into four clades, with the isolates obtained in this study clustering in CladeⅠ. Genetic diversity analysis revealed that Asian GPGV isolates exhibited high genetic diversity, potentially indicating an origin center, although population expansion occurred in Europe. This study represents the first comprehensive analysis of GPGV isolates from Inner Mongolia, providing critical insights into their genomic structure and evolutionary dynamics.