Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4326-4336.doi: 10.3864/j.issn.0578-1752.2021.20.007

• PLANT PROTECTION • Previous Articles     Next Articles

Molecular Variation and Phylogenetic Relationship of Apple Scar Skin Viroid in Seven Cultivars of Apple

LI ZiTeng,CAO YuHan,LI Nan,MENG XiangLong,HU TongLe,WANG ShuTong,WANG YaNan(),CAO KeQiang   

  1. College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
  • Received:2021-03-10 Accepted:2021-04-23 Online:2021-10-16 Published:2021-10-25
  • Contact: YaNan WANG E-mail:wyn3215347@163.com

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Abstract:

【Objective】The objective of this study is to explore the molecular variation and phylogenetic relationship of apple scar skin viroid (ASSVd) in different cultivars of apple, and to lay a foundation for further revealing the molecular variation mechanism of ASSVd. 【Method】The different cultivars of apple including Fuji, Tonami, Wang Lin, Mid-Autumn King, Gold Delicious, Xin Nonghong and Xin Nonghuang, infected by ASSVd were used as materials to amplify complete genome sequence by specific primers, and then molecular cloning and sequencing were performed. Biological software DNAMAN was used to analyze the identities of the variant sequences and the MEGA was used to construct a phylogenetic tree. 【Result】A total of 210 sequences of ASSVd were obtained, with a total of 17 variants, with a size of 325-333 nt. Seventeen variants of ASSVd were aligned and analyzed with other published representative isolates. The result showed that all the variants were divided into three groups. Group I includes 10 variants, which are closely related to the reported Baoding Fuji isolate (KR264032.1). Group II includes 6 variants, which are clustered separately. Group III includes one variant, which is closely related to the Xinjiang Fuji isolate (EU031455.1). Based on 8 variation base sites at genome 0-3, 221, 251, 284, and 302, the 17 variants were divided into 6 type including: 1. nt 0-3 (GGTA) + nt 41-46 (TAAAAT) + nt 221 (T) + nt 251 (T) +nt 284 (G) + nt 302 (T); 2. nt 0-3 (XGGT) + nt 41-46 (AGATAX) + nt 221 (T) + nt 251(X) + nt 284 (A) + nt 302 (A); 3. nt 0-3 (XGGT) + nt 41-46 (AGATAX) + nt 221 (X) + nt 251 (T) + nt 284 (A) + nt 302 (A); 4. nt 0-3 (XGGT/GGTA) + nt 41-46 (AGATAX) + nt 221 (T) + nt 251 (T) + nt 284 (A) + nt 302 (A); 5. nt 0-3 (GGTA) + nt 41-46 (TAAAAT) + nt 221 (X) + nt 251 (G) + nt 284 (G) + nt 302 (T); 6. nt 0-3 (GGTA) + nt 41-46 (TAAAAT) + nt 221 (T) + nt 251 (G) + nt 284 (G) + nt 302 (T). X represents missing. There are 6 types of variants in Fuji, type 4 (47.5%) is the main type. There are 3 types of variants in Wang Lin, type 5 (43%) is the main type. There are 3 types of variants in Gold Delicious, type 4 (60%) is the main type. ASSVd obtained from Tonami is type 5 (100%). ASSVd obtained from Mid-Autumn King is type 4 (100%). There are 2 types in Xin Nonghong, type 1 (83.3%) is the main type. There are 3 types in Xin Nonghuang, type 1 (62.5%) is the main type. 【Conclusion】Seventeen variants from 7 cultivars are divided into 6 types according to the 8 variation base sites of ASSVd nt 0-3, 221, 251, 284, and 302. Different apple cultivars carry different ASSVd population structures. The types and proportions of ASSVd variants are different.

Key words: apple scar skin viroid (ASSVd), genome, molecular cloning, molecular evolution, molecular variation

Fig. 1

The fruit symptom of different cultivars of apple infected by ASSVd a-f is the healthy fruit of Fuji, Tonami, Xin Nonghong, Wang Lin, Mid-Autumn King and Xin Nonghuang cultivars; A-F corresponds to the susceptible fruits of Fuji (color dappling), Tonami (color dappling), Xin Nonghong (color dappling), Wang Lin (russeting and distortion), Mid-Autumn King (russeting) and Xin Nonghuang (uneven and rough fruit surface)"

Fig. 2

The identity comparison among 17 variants of ASSVd from 7 cultivars of apple The specific information of each sequence is shown in Fig. 4. The same as Fig. 3"

Fig. 3

The cluster analysis between 17 ASSVd variants from 7 apple cultivars and other representative isolates"

Fig. 4

The phylogenetic relationship between 17 ASSVd variants from 7 apple cultivars and other representative isolates The phylogenetic tree was constructed with MEGA 7.0 by the neighbor-joining method, the Bootstrap was repeated 1 000 times. The description is represented as the isolate/origin and cultivar/accession number. The bootstrap values below 60% are not shown"

Fig. 5

Comparison of the sequences of all the isolates in this study with the ASSVd sequence from Baoding"

Table 1

Base types of ASSVd from different apple cultivars (%)"

苹果品种
Apple cultivar		 碱基类型Base type
Type 1 Type 2 Type 3 Type 4 Type 5 Type 6
富士Fuji 10.0 2.5 2.5 47.5 25.0 12.5
王林Wang Lin 28.4 0 0 0 43.0 28.6
金冠Gold Delicious 0 33.3 6.7 60.0 0 0
斗南Tonami 0 0 0 0 100.0 0
中秋王Mid-Autumn King 0 0 0 100.0 0 0
信侬红Xin Nonghong 83.3 0 0 0 0 16.7
信侬黄Xin Nonghuang 62.5 0 0 0 25.0 12.5

Table 2

Minimum free energy of secondary structure of different ASSVd isolates in this study"

来源
Source		 编号
Number		 长度
Length (nt)		 登录号
Accession number		 碱基类型
Base type		 最小自由能
Minimum free energy (kJ·mol-1)
斗南Tonami DN1 325 MH105019 5 -561.9
斗南Tonami DN1-2 331 MH105020 5 -583.2
富士Fuji FS1-1 329 MG891838 1 -563.1
富士Fuji FS4-1 330 MH105022 4 -565.6
富士Fuji FS4-9 329 MH105023 3 -567.7
富士Fuji FS4-13 329 MH105024 3 -567.7
富士Fuji FS3-4 326 MH105025 2 -558.1
富士Fuji FS3-10 329 MH105026 2 -575.7
王林Wang Lin WL1-5 332 MH105027 6 -568.1
王林Wang Lin WL1-6 331 MH105028 5 -583.2
王林Wang Lin WL2-2 333 MH105029 6 -577.8
王林Wang Lin WL3 328 MH105030 1 -558.1
信侬红Xin Nonghong Xnho1 332 MH105031 6 -576.1
信侬红Xin Nonghong Xnho1-3 332 MH105032 1 -579.5
信侬黄Xin Nonghuang Xnhu1-3 332 MH105033 6 -572.3
中秋王Mid-Autumn King ZQW7 331 MH105034 4 -563.5
金冠Gold Delicious GD3-23 329 MH105035 2 -575.7
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