Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (2): 266-280.doi: 10.3864/j.issn.0578-1752.2025.02.005

• PLANT PROTECTION • Previous Articles     Next Articles

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

PAN Yuan(), WANG De, LIU Nan, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan()   

  1. College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
  • Received:2024-08-28 Accepted:2024-10-12 Online:2025-01-21 Published:2025-01-21
  • Contact: WANG YaNan

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Abstract:

【Objective】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.【Method】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.【Result】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).【Conclusion】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.

Key words: macro-transcriptome sequencing, small RNA sequencing, apple virus, identification, Hebei Province

Table 1

Weight values of various indicators for sequencing data and splicing results"

指标
Index		 病毒contigs数量
Viral contigs number		 病毒contigs长度
Viral contigs length		 N50值
N50 value		 N90值
N90 value		 完整性
Completeness		 病毒种类
Viral type
权重Weight 0.0740 0.0740 0.1316 0.1316 0.2273 0.3615

Table 2

Scoring criteria of various indicators for sequencing data and splicing results"

指标
Index		 分值Score
2 4 6 8 10
病毒contigs数量Viral contigs number 0-12 12-24 24-36 36-48 48-50
病毒contigs长度Viral contigs length (bp) 1-1500 1500-3000 3000-4500 4500-6000 6000-7500
N50值N50 value (bp) 0-600 600-1200 1200-1800 1800-2400 2400-3000
N90值N90 value (bp) 0-100 100-200 200-300 300-400 400-500
完整性Completeness (%) 0-20 20-40 40-60 60-80 80-100
病毒种类Viral type 0-1 2-3 4-5 6-7 8

Table 3

Primers information used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 产物大小
Product size (bp)		 用途
Purpose		 参考文献
Reference
ASPV-F TGGAACCTCATGCTGCA 360 ASPV鉴定
ASPV identification		 [26]
ASPV-R TTGGGATCAACTTTACTAAAAAGCATAA
ARWV-F ACAAGGCAGTAGTTATTATCAGCAA 484 ARWV-2鉴定
ARWV-2 identification		 [19]
ARWV-R TTCTGCAACTAACTTCAAGGCTG
ASGV-F CCCGCTGTTGGATTTGATACACCTC 525 ASGV鉴定
ASGV identification		 [12]
ASGV-R CTGCAAGACCGCGACCAAGTTT
ACLSV-F GAGAGTTTCAGTTTGCTAGACA 566 ACLSV鉴定
ACLSV identification		 [27]
ACLSV-R GCAAATTCAGTCTGTAAAAG
AGCaV-F TCGGAAAGACTGTCCCGTTG 647 AGCaV鉴定
AGCaV identification		 [4]
AGCaV-R ACTCTTGCCCCCGATTCTTG
ApNMV-F ATGGTGTGCAATCGCTGTCA 657 ApNMV鉴定
ApNMV identification		 [28]
ApNMV-R CATCGACCATAAGGATATCA
CTLV-F CCCTCTCAGCTAGAATTGAA 889 CTLV鉴定
CTLV identification		 [29]
CTLV-R AGAGTGGACAAACTCTAGAC
CCGaV-F TGCCCATCCTTCTAACCTG 954 CCGaV鉴定
CCGaV identification		 [21]
CCGaV-R TTCCCATACAGTTTGCCG

Table 4

Information on representative isolates for ARWV-2 sequence identity analysis"

分离物
Isolate		 登录号
Accession number		 寄主
Host		 国家
Country
Fuji-BJ OP547336 苹果Apple 巴西Brazil
R12 MF062141 苹果Apple 德国Germany
LYXS MZ819711 梨Pear 中国China
982-11 MF062129 苹果Apple 加拿大Canada
982-11 MF062127 苹果Apple 加拿大Canada

Table 5

Information on representative isolates for CCGaV sequence identity analysis"

分离物
Isolate		 登录号
Accession number		 寄主
Host		 国家
Country		 分离物
Isolate		 登录号
Accession number		 寄主
Host		 国家
Country
Mishima MK940543 苹果Apple 巴西Brazil HU_Reglindis ON593790 苹果Apple 匈牙利Hungary
Gala MK940542 苹果Apple 巴西Brazil HU_Jonagold ON593789 苹果Apple 匈牙利Hungary
Fuji-BJ OP556109 苹果Apple 巴西Brazil CGW2 NC_035454 柑橘Citrus 意大利Italy
Weihai MZ926714 苹果Apple 中国China UN7 MH137741 柑橘Citrus 中国China
Gala-BJ OP820577 苹果Apple 巴西Brazil LR3 MG764564 柑橘Citrus 意大利Italy
AC1 MH038043 苹果Apple 美国USA HU_Florina ON593791 苹果Apple 匈牙利Hungary
CCGaV_RNA2_T OR640154 苹果Apple 土耳其Turkey CGW2 KX960111 柑橘Citrus 意大利Italy
CE-c3 OK495691 苹果Apple 意大利Italy

Fig. 1

The symptom of suspected apple virus sample for evaluation of high-throughput sequencing technique"

Table 6

Statistics of the sequencing data and assembly results of the macro-transcriptome sequencing and small RNA sequencing of apple fruit peel and branch bark"

样品种类
Sample type		 测序方法
Sequencing method		 Clean reads数量
Clean reads number		 Contigs
数量
Contigs number		 Contigs
长度
Contigs length (bp)		 N50值N50 value (bp) N90值N90 value (bp) 病毒来源reads数量
Viral source reads number		 病毒contigs数量
Viral contigs number		 病毒contigs
长度
Viral contigs length (bp)
果皮
Fruit peel		 宏转录组测序
Macro-transcriptome sequencing		 5146706 9161 300-7543 2620 451 58188 31 319-7506
小RNA测序
Small RNA sequencing		 9274079 2093 33-362 1150 282 144 12 213-2708
枝皮
Branch bark		 宏转录组测序
Macro-transcriptome sequencing		 7901932 12244 300-6470 1339 396 12738 60 304-6470
小RNA测序
Small RNA sequencing		 9896385 793 300-460 738 232 526 10 205-2726

Table 7

Statistics of viruses detected by macro-transcriptome sequencing and small RNA sequencing using apple fruit peel as samples"

测序方法
Sequencing method		 病毒名称
Virus name		 Reads数量
Reads number		 Contigs数量Contigs number 最长序列
Longest sequence (bp)		 最短序列
Shortest sequence (bp)		 覆盖深度
Sequencing depth		 完整性
Completeness (%)
宏转录组测序
Macro-transcriptome sequencing		 苹果坏死花叶病毒ApNMV 33754 3 3276 1882 770.00 98.95
苹果褪绿叶斑病毒ACLSV 17808 16 7506 319 48.00 99.40
苹果茎沟病毒ASGV 6926 6 6474 645 46.00 99.80
苹果茎痘病毒ASPV 150 6 944 321 7.00 97.20
小RNA测序
Small RNA sequencing		 苹果坏死花叶病毒ApNMV 96 2 1150 494 52.00 25.20
苹果褪绿叶斑病毒ACLSV 23 6 2708 1885 37.00 7.20
苹果茎沟病毒ASGV 3 2 957 552 2.00 12.10
苹果茎痘病毒ASPV 22 2 282 213 10.00 12.65

Table 8

Statistics of viruses detected by macro-transcriptome sequencing and small RNA sequencing using apple branch bark as samples"

测序方法
Sequencing method		 病毒名称
Virus name		 Reads数量Reads number Contigs数量Contigs number 最长序列
Longest sequence (bp)		 最短序列
Shortest sequence (bp)		 覆盖深度
Sequencing depth		 完整性
Completeness (%)
宏转录组测序
Macro-transcriptome sequencing		 苹果茎痘病毒ASPV 1550 30 1445 304 13.70 96.90
苹果茎沟病毒ASGV 5403 6 6470 393 41.97 97.60
苹果坏死花叶病毒ApNMV 1908 2 2617 1886 69.04 98.95
苹果绿皱果相关病毒AGCaV 18 1 323 323 7.01 98.95
柑橘囊胶相关病毒CCGaV 1112 5 2262 336 25.20 98.95
柑橘碎叶病毒CTLV 31 1 355 355 12.70 93.50
苹果褪绿叶斑病毒ACLSV 934 8 2474 400 14.03 98.80
苹果胶质木病毒-2 ARWV-2 1326 3 1339 371 49.74 98.95
小RNA测序
Small RNA sequencing		 苹果褪绿叶斑病毒ACLSV 40 6 1850 2300 3.63 1.32
苹果胶质木病毒-2 ARWV-2 301 2 1342 368 0.62 0.21
苹果坏死花叶病毒ApNMV 185 2 2726 2726 0.26 0.08

Table 9

Types of virus detected by macro-transcriptome sequencing using apple fruit peel and branch bark as samples"

检出病毒Virus 组织部位Tissue type 检出病毒Virus 组织部位Tissue type
苹果坏死花叶病毒ApNMV 果皮、枝皮Fruit peel, branch bark 苹果胶质木病毒-2 ARWV-2 枝皮Branch bark
苹果褪绿叶斑病毒ACLSV 果皮、枝皮Fruit peel, branch bark 苹果绿皱果相关病毒AGCaV 枝皮Branch bark
苹果茎沟病毒ASGV 果皮、枝皮Fruit peel, branch bark 柑橘囊胶相关病毒CCGaV 枝皮Branch bark
苹果茎痘病毒ASPV 果皮、枝皮Fruit peel, branch bark 柑橘碎叶病毒CTLV 枝皮Branch bark

Table 10

Types of virus detected by small RNA sequencing using apple fruit peel and branch bark as samples"

检出病毒Virus 组织部位Tissue type
苹果坏死花叶病毒ApNMV 果皮、枝皮Fruit peel, branch bark
苹果褪绿叶斑病毒ACLSV 果皮、枝皮Fruit peel, branch bark
苹果茎沟病毒ASGV 果皮Fruit peel
苹果茎痘病毒ASPV 果皮Fruit peel
苹果胶质木病毒-2 ARWV-2 枝皮Branch bark

Table 11

Comprehensive score of macro-transcriptome sequencing and small RNA sequencing of apple fruit peel and branch bark"

样品种类
Sample type		 测序方法
Sequencing method		 病毒contigs数量
Viral contigs number		 病毒contigs长度
Viral contigs length		 N50值
N50 value		 N90值
N90 value		 完整性
Completeness		 病毒种类
Viral type		 综合得分
Synthesis score
果皮
Fruit peel		 宏转录组测序
Macro-transcriptome sequencing		 0.2220 0.3700 0.6580 0.6580 1.1365 1.0845 4.1290
小RNA测序
Small RNA sequencing		 0.0740 0.1480 0.2632 0.3948 0.2273 1.0845 2.1918
枝皮
Branch bark		 宏转录组测序
Macro-transcriptome sequencing		 0.3700 0.3700 0.3948 0.5264 1.1365 1.8075 4.6052
小RNA测序
Small RNA sequencing		 0.0740 0.1480 0.2632 0.3948 0.2273 0.7230 1.8303

Fig. 2

Identification of viruses in apple samples by RT-PCR"

Fig. 3

Position of ARWV-2-HB contigs in the genome The sequence position is referenced to R12 (MF062141)"

Fig. 4

Position of CCGaV-HB contigs in the genome The sequence position is referenced to CGW2 (KX960111)"

Table 12

Sequence identity between the ARWV-2-HB isolate and representative isolates (%)"

分离物
Isolate		 登录号
Accession number		 RdRp (nt 2719-3089) CP (nt 84-941) MP (nt 12-379)
nt aa nt aa nt aa
Fuji-BJ OP547336 98.9 97.5 99.0 99.6 98.9 98.0
R12 MF062141 97.3 96.7 98.2 98.2 98.0 98.1
LYXS MZ819711 98.9 99.1 98.6 100.0 98.6 98.3
982-11 MF062129 97.5 96.7 98.4 98.9 - -

Table 13

Sequence identity between the CCGaV-HB isolate and representative isolates (%)"

分离物
Isolate		 登录号
Accession number		 RdRp CP (nt 49-1269) MP (nt 1590-2639)
nt 5-1532 nt 1937-4198 nt 4887-6647 nt aa nt aa
Mishima MK940543 99.8 99.7 99.7 99.9 100.0 100.0 100.0
Gala MK940542 100.0 99.7 99.8 99.8 99.7 100.0 100.0
Fuji-BJ OP556109 99.9 99.6 99.7 99.9 100.0 99.8 99.5
Weihai MZ926714 99.7 99.6 99.4 99.6 99.4 99.6 99.5
Gala-BJ OP820577 - 99.7 - 99.9 100.0 99.9 100.0
AC1 MH038043 98.7 99.1 98.2 99.3 99.7 99.0 99.0
CCGaV_RNA2_T OR640154 99.8 99.6 99.7 99.3 99.7 98.6 98.2
CE-c3 OK495691 98.6 98.7 98.4 99.3 99.7 98.5 98.0
HU_Reglindis ON593790 - 99.4 99.6 99.1 99.4 98.8 98.5
HU_Jonagold ON593789 98.7 - - 98.8 99.1 98.3 98.5
CGW2 NC_035454 96.3 97.2 97.0 97.5 98.5 97.6 96.8
UN7 MH137741 96.5 97.3 96.4 97.6 98.5 97.6 96.3
LR3 MG764564 96.3 97.3 97.0 97.5 98.5 97.5 96.3
HU_Florina ON593791 99.3 - - 99.5 99.7 99.6 99.7
CGW2 KX960111 96.3 97.2 97.0 97.5 98.5 97.6 100.0

Fig. 5

Phylogenetic tree of ARWV-2-HB isolate and other ARWV representative isolates based on the RNASb CP amino acid sequence"

Fig. 6

Phylogenetic tree of CCGaV-HB isolate and other CCGaV representative isolates based on the CP amino acid sequence"

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