侵染牡丹的苹果茎沟病毒分离物基因组测序及分析

doi:10.16420/j.issn.0513-353x.2023-0277

摘要/Abstract

摘要：

前期在北京地区种植的牡丹中检测到了苹果茎沟病毒（apple stem grooving virus，ASGV），并获得了1个ASGV牡丹分离物的基因组全序列。为分析侵染牡丹的ASGV的遗传多样性，根据已报道的118个ASGV基因组全序列的保守区设计特异性引物，用重叠（overlapping）RT-PCR和cDNA末端序列快速扩增（rapid amplification of cDNA ends，RACE）技术获得9个ASGV牡丹分离物的基因组全序列。共计10个ASGV牡丹分离物，基因组全序列成对比对的核苷酸一致性为81.9% ~ 97.6%，与其他物种ASGV分离物基因组全序列的核苷酸一致性为80.8% ~ 91.2%。序列分析表明ASGV牡丹分离物的分子多样性主要位于多聚蛋白的保守结构域Mtr和P-Pro之间以及RdRp与CP之间。用127个ASGV基因组全序列进行重组分析，5个牡丹分离物被鉴定为重组体，其中1个重组体的重组亲本来源于不同国家的不同寄主植物。用79个非重组的ASGV基因组全序列构建系统发育树，表明ASGV牡丹分离物与ASGV苹果、梨和柑橘等分离物共同聚类到第Ⅰ组，且ASGV牡丹分离物聚到不同的分支。研究结果表明侵染牡丹的ASGV具有遗传多样性，重组可能是导致其遗传多样性的一个因素。

关键词: 苹果茎沟病毒, 牡丹, 基因组全序列, 序列分析, 重组, 系统发育

Abstract:

Apple stem grooving virus（ASGV）was detected in tree peony（Paeonia sect. moutan）plants cultivated in Beijing region in previous study，and the complete genome sequence of one ASGV-tree peony isolate was obtained. To analyze the genetic diversity of tree peony-infecting ASGV，the complete genome sequences of nine ASGV-tree peony isolates were obtained using overlapping RT-PCR and rapid amplification of cDNA ends（RACE）techniques with specific primers designed according to the conserved regions of 118 reported ASGV complete genome sequences. Pairwise alignment of the complete genome sequences of total ten ASGV-tree peony isolates showed the nucleotide identity of 81.9%-97.6%. Alignment of ASGV-tree peony isolates and other isolates showed the nucleotide identity of 80.8%-91.2%. Sequence analyses indicated that the molecular diversity of ASGV-tree peony isolates mainly located in the regions between Mtr and P-Pro，RdRp and CP，which were the conserved domains of ASGV-encoded polyprotein. Five ASGV-tree peony isolates were identified as recombinants，including one recombinant whose recombinant parents developed from different host plants in different countries，performed by recombination analyses using the complete genome sequences of 127 ASGV isolates. Phylogenetic tree constructed using 79 non-recombinant ASGV complete genome sequences suggested that ASGV-tree peony isolates clustered into the first group closely with ASGV isolates from apple，pear，citrus and so on，and ASGV-tree peony isolates clustered into different clades. These results revealed the genetic diversity of tree peony-infecting ASGV，and recombination is a factor that likely contributes to this diversity.

Key words: apple stem grooving virus, tree peony, complete genome sequence, sequence analyses, recombination, phylogeny

陈玲, 郭铖, 贾安宁, 邓丛良, 种焱, 史喜菊, 李永强. 侵染牡丹的苹果茎沟病毒分离物基因组测序及分析[J]. 园艺学报, 2023, 50(12): 2735-2747.

CHEN Ling, GUO Cheng, JIA Anning, DENG Congliang, CHONG Yan, SHI Xiju, LI Yongqiang. Determination and Analyses of the Complete Genome Sequences of Apple Stem Grooving Virus Isolates Infecting Tree Peony[J]. Acta Horticulturae Sinica, 2023, 50(12): 2735-2747.

图/表 10

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产物/bp Product	引物位置/nt Location	引物名称 Primer name	引物序列（5′-3′） Primer sequence
1 953	494 ~ 513	ASGV-F1	TGGTCTGAGGAACCAATGCC
	2 446 ~ 2 427	ASGV-R1	ATCCTCTTGACGTTTGGCGT
2 342	1 533 ~ 1 552	ASGV-F2	TCAAAGCTGGTCAGAGCGAG
	3 874 ~ 3 855	ASGV-R2	AAAGCATGGTCGAGCTTCCA
2 083	3 498 ~ 3 517	ASGV-F3	TCCATACTCGGCCTCTCCAA
	5 580 ~ 5 561	ASGV-R3	TTGGAGTTTGGCCCCCTTTT
	1 413 ~ 1 392	5ʹ RACE	GATTACGCCAAGCTT-AACCTGCGGCCAAGCAGGTCCAA
	5 128 ~ 5 152	3ʹ RACE	GATTACGCCAAGCTT-TGCCTGTTTGGACCCAGAAAGGGGT

产物/bp Product	引物位置/nt Location	引物名称 Primer name	引物序列（5′-3′） Primer sequence
1 953	494 ~ 513	ASGV-F1	TGGTCTGAGGAACCAATGCC
	2 446 ~ 2 427	ASGV-R1	ATCCTCTTGACGTTTGGCGT
2 342	1 533 ~ 1 552	ASGV-F2	TCAAAGCTGGTCAGAGCGAG
	3 874 ~ 3 855	ASGV-R2	AAAGCATGGTCGAGCTTCCA
2 083	3 498 ~ 3 517	ASGV-F3	TCCATACTCGGCCTCTCCAA
	5 580 ~ 5 561	ASGV-R3	TTGGAGTTTGGCCCCCTTTT
	1 413 ~ 1 392	5ʹ RACE	GATTACGCCAAGCTT-AACCTGCGGCCAAGCAGGTCCAA
	5 128 ~ 5 152	3ʹ RACE	GATTACGCCAAGCTT-TGCCTGTTTGGACCCAGAAAGGGGT

取样位置 Sampling location	植株症状 Plant symptom	分离物 Isolate	基因组大小/nt Genome size	登录号 Accession No.
海淀公园Haidian Park	褪绿Chlorosis	Peony BJ2	6 470	OQ658829
	卷叶Leafroll	Peony BJ3	6 470	OQ658830
景山公园Jingshan Park	褪绿Chlorosis	Peony BJ4	6 469	OQ658831
	皱缩、节间缩短Crinkle，internode shortening	Peony BJ5	6 467	OQ658832
	卷叶Leafroll	Peony BJ6	6 467	OQ658833
北京植物园Beijing Botanical Garden	红叶Leaf reddening	Peony BJ7	6 495	OQ658834
	卷叶Leafroll	Peony BJ8	6 495	OQ658835
圆明园Yuanmingyuan Garden	褪绿Chlorosis	Peony BJ9	6 469	OQ658836
	皱缩Crinkle	Peony BJ10	6 469	OQ658837

取样位置 Sampling location	植株症状 Plant symptom	分离物 Isolate	基因组大小/nt Genome size	登录号 Accession No.
海淀公园Haidian Park	褪绿Chlorosis	Peony BJ2	6 470	OQ658829
	卷叶Leafroll	Peony BJ3	6 470	OQ658830
景山公园Jingshan Park	褪绿Chlorosis	Peony BJ4	6 469	OQ658831
	皱缩、节间缩短Crinkle，internode shortening	Peony BJ5	6 467	OQ658832
	卷叶Leafroll	Peony BJ6	6 467	OQ658833
北京植物园Beijing Botanical Garden	红叶Leaf reddening	Peony BJ7	6 495	OQ658834
	卷叶Leafroll	Peony BJ8	6 495	OQ658835
圆明园Yuanmingyuan Garden	褪绿Chlorosis	Peony BJ9	6 469	OQ658836
	皱缩Crinkle	Peony BJ10	6 469	OQ658837

分离物Isolate	Peony BJ2	Peony BJ3	Peony BJ4	Peony BJ5	Peony BJ6	Peony BJ	Peony BJ7	Peony BJ8	Peony BJ9
Peony BJ3	96.4
Peony BJ4	87.4	87.6
Peony BJ5	87.4	86.8	92.2
Peony BJ6	83.3	83.5	90.3	97.0
Peony BJ	82.1	82.1	90.2	87.3	88.7
Peony BJ7	83.2	83.1	83.0	87.4	86.5	87.3
Peony BJ8	82.3	82.2	83.6	85.5	86.8	88.6	91.6
Peony BJ9	94.8	95.2	87.0	86.4	83.5	81.9	83.1	82.2
Peony BJ10	94.8	95.1	86.8	86.1	83.2	82.0	83.0	82.2	97.6
其他Other	82.4 ~ 912	82.3 ~ 91.3	81.9 ~ 86.8	81.8 ~ 87.2	82.6 ~ 87.9	81.2 ~ 89.0	82.1 ~ 90.5	80.8 ~ 91.0	82.0 ~ 90.7