甘薯羽状斑驳病毒O株系和RC株系中国分离物全基因组 序列分析及其遗传特征

doi:10.3864/j.issn.0578-1752.2020.11.007

Abstract

Abstract:

【Objective】The objective of this study is to clone the complete nucleotide sequence of Chinese isolate of Sweet potato feathery mottle virus (SPFMV) O and RC strains, elucidate the genomic structural characterization and variation of SPFMV-O-Ch1 and SPFMV-RC-Ch1, and to lay a foundation for the study of pathogenic mechanism of SPFMV. 【Method】According to the SPFMV genome sequences available in GenBank database, 2 pairs of degenerate primers and 3 pairs of specific primers were designed, the whole genome of SPFMV O and RC strains isolated from China was amplified by RT-PCR from sweet potato leaves infection with SPFMV and subsequently cloned into vector pMD19-T and sequenced. The complete genome sequences of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates were assembled by using DNAMAN. Genetic variation analyses of complete genomic sequences, polyproteins, and individual protein sequences were performed using DNAMAN. Neighbor-joining phylogenetic tree of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates with other isolates was constructed using MEGA7.0 software. Recombination analyses were carried out using RDP software. 【Result】The amplification and sequencing revealed that the complete nucleotide sequence of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates was 10 992 nucleotides (nt) and 10 851 nt in length, respectively. The viral genome of SPFMV-O-Ch1 isolate contained a single open reading frame (ORF) of 10 557 nt encoding a polyprotein of 3 518 aa. SPFMV-RC-Ch1 isolate polyprotein consisted of 10 482 nt and encoded 3 493 aa. Two small ORFs, P1N-PISPO and P3N-PIPO were identified in the P1 and P3 proteins of these two isolates. Pairwise comparisons of the complete genome nucleotide sequence showed that O-Ch1 had 87.3% identity with RC-Ch1 isolate and shared 86.0%-95.8% sequence identity with other SPFMV isolates. It was most closely related to the isolate Ruk73 with 95.8% nt identity and lowest nt identity with 11-1 isolate (86.0%). RC-Ch1 and other SPFMV isolates shared 85.9%-98.7% sequence identity at the complete genome nucleotide sequence level. It had the highest nt identity with IS90 isolate (98.7%), and had lowest nt identity with Aus1-2B isolate (85.9%). Phylogenetic tree analysis based on polyprotein gene indicated that SPFMV-O-Ch1 formed a branch with the isolates of O strain containing Ordinary, 10-O and 17-O, and SPFMV-RC-Ch1 formed a branch with the isolates of RC strain containing S, IS90 and CW137. Recombination analysis showed that there were three potential significant recombination events occurred in 7 731- 9 710, 135-10 012 and 4 825-6 948 nt of O-Ch1 isolate genome, respectively. No recombination event was detected in the complete genome of RC-Ch1 isolate. 【Conclusion】The genomic organizations of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates were same to other isolates. O-Ch1 isolate was closely related to the isolates of O strain and RC-Ch1 isolate was closely related to those isolates of RC strain. Three recombination events were detected in O-Ch1 isolate, but no recombination event was detected in RC-Ch1 isolate.

Key words: Sweet potato feathery mottle virus (SPFMV), strain, complete nucleotide sequence, genetic variation, recombination analysis

QIN YanHong,WANG YongJiang,WANG Shuang,QIAO Qi,TIAN YuTing,ZHANG DeSheng,ZHANG ZhenChen. Complete Nucleotide Sequence Analysis and Genetic Characterization of the Sweet potato feathery mottle virus O and RC Strains Isolated from China[J].Scientia Agricultura Sinica, 2020, 53(11): 2207-2218.

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Figures/Tables 7

Table 1

Name, strain, origin and accession number of all isolates used in this study"

序号Number	分离物名称Isolate name	株系Strain	地理来源Geographic origin	登录号Accession number
1	RC-Ch1	RC	中国China	KY296451
2	O-Ch1	O	中国China	KY296450
3	CW137	RC	韩国South Korea	KP115608
4	IS90	RC	韩国South Korea	KP115610
5	GJ122	O	韩国South Korea	KP115609
6	S	RC	日本Japan	D86371
7	10-O	O	日本Japan	AB439206
8	Ordinary	O	日本Japan	AB465608
9	17-O	O	日本Japan	AB509454
10	11-8	RC	美国USA	MH782223
11	95-204R	RC	美国USA	MH782224
12	11-1	RC	美国USA	MH778648
13	TFSW-1J	RC	美国USA	MH782227
14	95-2T	O	美国USA	MH782225
15	Aus7D	RC	澳大利亚Australia	MF572046
16	Aus12D	RC	澳大利亚Australia	MF572049
17	Aus8-8CA	RC	澳大利亚Australia	MG656422
18	Aus11D	RC	澳大利亚Australia	MF572048
19	Aus15-21CA	RC	澳大利亚Australia	MG656429
20	Aus11-13CA	RC	澳大利亚Australia	MG656425
21	Aus6-3B	RC	澳大利亚Australia	MG656420
22	Aus9-9B-2	RC	澳大利亚Australia	MG656423
23	Aus10-10D	RC	澳大利亚Australia	MG656424
24	Aus13-18B-1	RC	澳大利亚Australia	MG656427
25	Aus4D	RC	澳大利亚Australia	MF572047
26	Aus3D	RC	澳大利亚Australia	MF572052
27	Aus7-7B	RC	澳大利亚Australia	MG656421
28	Aus2D	RC	澳大利亚Australia	MF572051
29	Aus4-3A	RC	澳大利亚Australia	MG656418
30	Aus9D	RC	澳大利亚Australia	MF572054
31	Aus14-20CA	RC	澳大利亚Australia	MG656428
32	Aus12-16B	RC	澳大利亚Australia	MG656426
33	Aus16-24A	RC	澳大利亚Australia	MG656430
34	Aus3-2BC	RC	澳大利亚Australia	MG656417
35	Aus17-11D	O	澳大利亚Australia	MG656431
36	Aus5-3B	O	澳大利亚Australia	MG656419
37	Aus13B	O	澳大利亚Australia	MF572050
38	Aus1-2B	O	澳大利亚Australia	MG656415
39	Aus2-2BC	O	澳大利亚Australia	MG656416
40	UNB-01	RC	巴西Brazil	MF185715
41	TM33C	O	东帝汶East Timor	MF572055
42	TM64B	O	东帝汶East Timor	MF572053
43	TM66B	O	东帝汶East Timor	MF572056
44	Ruk73	O	乌干达Uganda	KP729265
45	RC-Arg	RC	阿根廷Argentina	KF386014
46	O-Arg	O	阿根廷Argentina	KF386013
47	SRF109a	O	肯尼亚Kenya	MH264535
48	ZA-O	O	南非South Africa	KT069222
49	Piu3	EA	秘鲁Peru	FJ155666
50	AM-MB2		西班牙Spain	KU511268

Table 1

Table 2

Fig. 1

Table 3

Fig. 2

Fig. 3

Table 4

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扩增片段 Amplified fragment	引物名称 Primer name	引物序列 Primer sequence (5′→3′)	目的片段大小 Expected size (bp)
O-1/RC-1	1-F	AATAACACAACWCAAYACAACAYAASAAAACT	4784/4711
O-1/RC-1	4711-R	GTGATATCATCTAARCTYTGATG	4784/4711
O-2	4689-F	CATCARAGYTTAGATGATATCAC	5692
O-2	10380-R	CATATCGCGCAAGACTCATATC	5692
O-3	SPFMV-O-4160-F	AAGCGACAGAGCAACAGACTTATGTACTA	1124
O-3	SPFMV-O-5284-R	GATATTGCGTTGTAAATTCAACCTCACGTC	1124
RC-2	SPFMV-RC-4260-F	AAGTTTGTGAATATGCTACTAGTCATCA	3814
RC-2	SPFMV-RC-8220-R	TACCTTCTAGGTCTGTGATCAGTTTAGAC	3814
RC-3	SPFMV-RC-7580-F	CCGATAGCTAGCGTCATATGCCATCTCG	3426
RC-3	SPFMV-11000-R	TTGGCTCGATCACGAACCAAAAAGGCT	3426

基因组区域 Genome region	O-Ch1蛋白大小及与48个分离物比对 Protein size of O-Ch1 and comparison with 48 isolates			RC-Ch1蛋白大小及与48个分离物比对 Protein size of RC-Ch1 and comparison with 48 isolates			O-Ch1与RC-Ch1比对 Comparison of O-Ch1 and RC-Ch1
基因组区域 Genome region	蛋白大小（氨基酸） Protein size (aa)	核苷酸一致性 nt identity (%)	氨基酸一致性 aa identity (%)	蛋白大小（氨基酸） Protein size (aa)	核苷酸一致性 nt identity (%)	氨基酸一致性 aa identity (%)	核苷酸（氨基酸）一致性 nt (aa) identity (%)
全长Complete sequence	-	86.0-95.8	-	-	85.9-98.7	-	87.3 (-)
多聚蛋白Polyprotein	3518	87.0-95.8	92.1-97.6	3493	87.0-98.8	92.8-99.3	88.2 (94.0)
P1	689	83.4-97.8	81.2-96.4	664	82.8-99.0	80.6-99.2	85.4 (83.9)
HC	458	83.3-98.2	91.9-98.9	458	83.4-99.0	92.1-100.0	84.0 (93.2)
P3	352	93.2-98.5	95.5-98.6	352	92.8-99.3	94.3-99.4	93.8 (96.0)
6K1	52	92.9-100.0	92.3-100.0	52	91.7-98.7	94.2-100.0	94.2 (98.1)
CI	643	89.0-98.0	97.0-99.7	643	89.7-98.9	97.5-99.8	89.9 (98.0)
6K2	53	91.2-98.7	94.3-100.0	53	89.3-98.7	96.2-100.0	91.8 (98.1)
NIa-VPg	192	78.1-97.7	90.6-100.0	192	83.9-99.3	89.6-100.0	84.4 (97.4)
NIa-Pro	243	83.4-96.3	93.4-98.8	243	79.0-98.6	92.6-99.6	85.9 (96.3)
NIb	521	86.3-97.3	93.1-98.5	521	85.3-98.7	92.9-99.8	89.5 (96.2)
CP	315	76.7-96.6	94.0-99.7	315	79.9-98.9	95.6-99.7	92.5 (96.8)

序号 Number	主要亲本 Major parent	次要亲本 Minor parent	位置 Position (nt)	P值P-Value
序号 Number	主要亲本 Major parent	次要亲本 Minor parent	位置 Position (nt)	RDP	GENECONV	BootScan	MaxChi	Chimaera	SiScan	3Seq
1	Ordinary- AB465608	TM33C- MF572055	7731- 9710	5.583×10^-33	8.317×10^-17	7.407×10^-31	3.758×10^-20	3.466×10^-22	2.430×10^-25	1.095×10^-44
2	EA-Piu3- FJ155666	Aus5-3B- MG656419	135- 10012	-	-	-	8.783×10^-4	7.437×10^-3	1.803×10^-20	1.561×10^-56
3	Aus13B- MF572050	EA-Piu3- FJ155666	4825- 6948	-	-	-	1.104×10^-3	1.071×10^-2	-	1.256×10^-2

Complete Nucleotide Sequence Analysis and Genetic Characterization of the Sweet potato feathery mottle virus O and RC Strains Isolated from China

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