Evaluation of the Effectiveness of Two High-Throughput Sequencing Techniques in Identifying Apple Viruses and Identification of Two Novel Viruses

Macro-transcriptome sequencing and small RNA sequencing are commonly used high-throughput sequencing techniques in virus identification. The objective of this study is to explore the application efficiency of macro-transcriptome sequencing and small RNA sequencing in the identification of emerging viruses in apples, analyze the impact of different tissue types on the identification results, and to provide a basis for the accurate diagnosis of apple virus diseases.

The samples of apple peel and branch bark were collected from ‘Luli’ apple trees exhibiting novel viral symptoms in Shenzhou County of Hengshui City in August 2022. Total RNA was extracted, and macro-transcriptome libraries and small RNA libraries were constructed for high-throughput sequencing. Bioinformatic techniques and software were utilized to analyze and evaluate the sequencing data. Initially, the indicators from the high-throughput sequencing technique results were compared. Subsequently, a comprehensive evaluation of the effectiveness of each sequencing method was conducted using the analytic hierarchy process (AHP) and a 5-level grading system to calculate the weighted values of these indicators. Finally, RT-PCR was employed to validate the high-throughput sequencing results, and the genomic characteristics and phylogenetic relationships of the emerging viruses were analyzed.

In terms of splicing effect, using the same tissue material, the macro-transcriptome sequencing outperformed small RNA sequencing. When the same technique was applied, the splicing effect for fruit peel tissue was better than that of branch bark. In terms of the number of virus species detected, macro-transcriptome sequencing identified the highest number of virus species in branch bark, including eight viruses: apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple necrotic mosaic virus (ApNMV), apple rubbery wood virus 2 (ARWV-2), apple green crinkle associated virus (AGCaV), citrus concave gum-associated virus (CCGaV), and citrus tatter leaf virus (CTLV). In contrast, small RNA sequencing technique detected the fewest virus types in branch bark. There were differences in virus types between fruit peels and branch barks detected through small RNA sequencing technique. When fruit peels were used as the detection target, both methods identified the same number of virus types. After comprehensively comparing the synthesis score of various indicators, the macro-transcriptome sequencing of bark samples scored the highest. The results of high-throughput sequencing were consistent with those obtained through RT-PCR. ARWV-2 and CCGaV were discovered for the first time in Hebei Province, and were designated as ARWV-2-HB and CCGaV-HB. The GenBank accession numbers for the coat protein (CP) gene of ARWV-2-HB and the movement protein (MP) and CP genes of CCGaV-HB are PQ095583, PQ095581, and PQ095582. The genomic sequences of both viruses showed over 96% identity to their respective representative isolates. Phylogenetic trees constructed based on the CP amino acid sequences of ARWV-2 and CCGaV revealed that ARWV-2-HB was most closely related to LYXS (MZ819711), while CCGaV-HB exhibited relatively close relationships with Mishima (MK940543), Gala (MK940542), Gala-BJ (OP820577), Fuji-BJ (OP556109), and AC1 (MH038043).

Using macro-transcriptome sequencing and small RNA sequencing techniques, the fruit peel and branch bark of the same ‘Luli’ apple tree were sequenced separately. Among two sequencing methods, the macro-transcriptome sequencing of branch bark showed the best sequencing effect, discovered the highest number of viruses and relatively complete viral genome sequences. When using small RNA sequencing, only a portion of virus types could be detected in both fruit peels and branch barks. Due to the differences in virus types detected from different tissue materials, it is recommended to test both tissue materials simultaneously. ARWV-2 and CCGaV were reported in Hebei Province in this study, and partial genomic sequences of ARWV-2-HB and CCGaV-HB were revealed, which enriching the genomic sequence information of ARWV-2 and CCGaV. Furthermore, the phylogenetic relationships of these two viruses with other representative isolates have been clarified.