苹果锈果类病毒在7个品种苹果上的分子变异及系统发育关系

doi:10.3864/j.issn.0578-1752.2021.20.007

摘要/Abstract

摘要：

【目的】探究不同品种苹果锈果类病毒（apple scar skin viroid,ASSVd）的分子变异及系统发育关系,为进一步揭示ASSVd分子变异机制打下基础。【方法】以富士、斗南、王林、中秋王、金冠、信侬红和信侬黄7个品种的染病苹果组织为材料,通过特异性引物对ASSVd全基因组序列进行扩增,然后进行分子克隆和序列测定,利用生物学软件DNAMAN对变异序列一致性进行分析并利用生物学软件MEGA构建系统发育树。【结果】共获得210个ASSVd的基因组序列,共计17种变体,大小为325—333 nt。将不同苹果品种获得的17种变体与其他已发表代表性分离物进行分析发现,所有变体被分为3组,组Ⅰ包括10种变体,同已报道的保定富士分离物（KR264032.1）亲缘关系较近;组Ⅱ包括6种变体单独聚类;组Ⅲ包括1种变体,与新疆红富士苹果上的分离物（EU031455.1）亲缘关系较近。进一步根据基因组0—3、221、251、284、302这8个位点的碱基变异,将17种变体分为6种类型：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）。其中X表示缺失。富士品种包含6种变体,以类型4为主（47.5%）;王林品种包含3种变体,以类型5为主（43%）;金冠包含3种变体,以类型4为主（60%）;斗南品种只有1种变体,为类型5,占比100%;中秋王品种只有1种变体,为类型4,占比100%;信侬红包含2种变体,以类型1为主（83.3%）;信侬黄包含3种变体,以类型1为主（62.5%）。【结论】根据ASSVd nt 0—3、221、251、284、302这8个位点的碱基变异,将富士、斗南、王林、中秋王、金冠、信侬红、信侬黄品种中17种变体分为6种类型,不同苹果品种携带的ASSVd种群结构、病毒变体类型及占比均不同。

关键词: 苹果锈果类病毒, 基因组, 分子克隆, 分子进化, 分子变异

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

李紫腾,曹钰晗,李楠,孟祥龙,胡同乐,王树桐,王亚南,曹克强. 苹果锈果类病毒在7个品种苹果上的分子变异及系统发育关系[J]. 中国农业科学, 2021, 54(20): 4326-4336.

LI ZiTeng,CAO YuHan,LI Nan,MENG XiangLong,HU TongLe,WANG ShuTong,WANG YaNan,CAO KeQiang. Molecular Variation and Phylogenetic Relationship of Apple Scar Skin Viroid in Seven Cultivars of Apple[J]. Scientia Agricultura Sinica, 2021, 54(20): 4326-4336.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.20.007

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苹果品种 Apple cultivar	碱基类型Base type
苹果品种 Apple cultivar	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

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