广西辣椒病毒的sRNA深度测序和RT-PCR鉴定

doi:10.16420/j.issn.0513-353x.2021-0673

摘要/Abstract

摘要：

采用小RNA（sRNA）深度测序技术,结合RT-PCR技术对广西9个辣椒主产区具有典型病毒病症状的病样进行检测,共发现10种病毒,其中小米椒内源RNA病毒1（Capsicum frutescens endornavirus 1,CFEV 1）、辣椒潜隐病毒2（pepper cryptic virus 2,PCV 2）、烟草轻型绿花叶病毒（tobacco mild green mosaic virus,TMGMV）和辣椒黄脉病毒（pepper vein yellows virus,PeVYV）是在广西辣椒上首次发现,且CFEV 1在中国是首次发现。10种病毒在75份辣椒病样中的总检出率为8.00% ~ 66.67%,前5位依次为辣椒脉斑驳病毒（chilli veinal mottle virus,ChiVMV,66.67%）、甜椒脉斑驳病毒（pepper veinal mottle virus,PVMV,62.67%）、黄瓜花叶病毒（cucumber mosaic virus,CMV,41.33%）、CFEV 1（32.0%）以及PCV 2（26.67%）。ChiVMV、PVMV、CMV和CFEV 1在广西7 ~ 9个辣椒主产区广泛分布,推测为优势病毒。除桂林外,其他8个辣椒主产区均含以上4种优势病毒中的1 ~ 3种,但在桂林,这4种病毒的检出率偏低或检不出,其优势病毒种类是PMMoV和PCV 2。广西辣椒病毒病复合侵染率为94.66%,3种（检出率42.67%）和4种（检出率24.00%）病毒复合侵染是主要形式。3种复合侵染中以ChiVMV + PVMV + CMV和ChiVMV + CFEV 1 + ChiRSV占比最高（均为18.75%）,4种复合侵染中以ChiVMV + PVMV + CMV + CFEV 1占比最高（16.67%）。

关键词: 辣椒, 病毒病, 小RNA, 深度测序, RT-PCR, 分子鉴定

Abstract:

We analysed diseased pepper samples with typical viral symptoms collected from nine of major pepper growing areas in Guangxi through small RNA（sRNA）deep sequencing technology and RT-PCR. The results showed that ten viruses were identified,among which,Capsicum frutescens endornavirus 1（CFEV 1）,pepper cryptic virus 2（PCV 2）,tobacco mild green mosaic virus（TMGMV）and pepper vein yellows virus（PeVYV）were detected on pepper for the first time in Guangxi,and CFEV 1 was also detected for the first time in China. The total detection rates of ten viruses in 75 diseased pepper samples were 8.00%-66.67%. The top five viruses were chilli veinal mottle virus（ChiVMV,66.67%）,pepper veinal mottle virus（PVMV,62.67%）,cucumber mosaic virus（CMV,41.33%）,CFEV 1（32.0%）and PCV 2（26.67%）. ChiVMV,PVMV,CMV and CFEV 1 were found to be widely distributed across seven to nine of the major pepper growing areas of Guangxi,which were speculated to be the dominant viruses infecting peppers in Guangxi. Except Guilin,other eight regions contained at least one to three of the four dominant viruses. However,in Guilin,the detection rates of these four dominant viruses were relatively low or negligible,and the dominant viruses were PMMoV and PCV 2. The mixed infection rate of pepper virus disease was up to 94.66% in Guangxi,and the mixed infection were mostly caused by a complex of three（detection rate 42.67%）or four viruses（detection rate 24.00%）in Guangxi. Mixed infection of three viruses were predominantly caused by ChiVMV + PVMV + CMV and ChiVMV + CFEV 1 + ChiRSV both with detection rate of 18.75%,that of four viruses was mostly caused by ChiVMV + PVMV + CMV + CFEV 1 with detection rate of 16.67%.

Key words: pepper, viral diseases, small RNA, deep sequencing, RT-PCR, molecular detection

中图分类号:

S641.3

龚明霞, 赵虎, 王萌, 吴星, 赵曾菁, 何志, 黄金梅, 孟生德, 王日升. 广西辣椒病毒的sRNA深度测序和RT-PCR鉴定[J]. 园艺学报, 2022, 49(5): 1060-1072.

GONG Mingxia, ZHAO Hu, WANG Meng, WU Xing, ZHAO Zengjing, HE Zhi, HUANG Jinmei, MENG Shengde, WANG Risheng. Identification of Viruses Infecting Peppers in Guangxi by Small RNA Deep Sequencing and RT-PCR[J]. Acta Horticulturae Sinica, 2022, 49(5): 1060-1072.

图/表 9

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病毒 Virus	引物序列（5′-3′） Sequence	片段大小/bp Product	退火温度/℃ T_m
ChiVMV	F：ATAATTTGTCCCAACCACCTGA;R：CTCACAAGCATTAACACAGAGC	923	51
CMV	F：GGTTCTGGAAAACACAGGTTCA;R：GAGTCATGGACAAATCTGAATCAAC	682	56
PVMV	F：AGGTGTGTAAATGATTTCGGC;R：GAAAAAGTGAGAGCCAGTTAGT	825	50
PMMoV	F：TCCGAGAAGTGCCGACA;R：TCAATTTGTCTGCCGCTGAG	992	52
ChiRSV	F：ATGAACTGTACGCCGAAGGG;R：AGTCCGATCGCCTATGAGGA	897	56
PeVYV	F：GACGACGAAATGGAGGC;R：CATTAATCTGCGAAGCCCG	361	51
CFEV 1	F：TGTGCGTGAAGGGTGTTTCT;R：GCGGTCATGCGAGTAGTGAA	568	56
PCV 2	F：CACGTGTGAGGGCATTTGAC;R：GTCCGACACCGTATGCCTTT	717	57
TMGMV	F：TTCCGCTTATGCAGATCCTGT;R：GACGAACAACCACTGCTGAT	523	54
PepMoV	F：GGACTTCTGTTTGTCGGTGAT;R：AGATGTCTGAAGTGAGTCATCAG	385	50
PVY	F：AGCAAAGCAGCATCCAGTCA;R：GTACTGATGCCACCGTCCAA	670	56
TMV	F：GTTCTTGTCATCTGCGTGGG;R：GCTCTCGAAAGAGCTCCGAT	406	57

病毒 Virus	引物序列（5′-3′） Sequence	片段大小/bp Product	退火温度/℃ T_m
ChiVMV	F：ATAATTTGTCCCAACCACCTGA;R：CTCACAAGCATTAACACAGAGC	923	51
CMV	F：GGTTCTGGAAAACACAGGTTCA;R：GAGTCATGGACAAATCTGAATCAAC	682	56
PVMV	F：AGGTGTGTAAATGATTTCGGC;R：GAAAAAGTGAGAGCCAGTTAGT	825	50
PMMoV	F：TCCGAGAAGTGCCGACA;R：TCAATTTGTCTGCCGCTGAG	992	52
ChiRSV	F：ATGAACTGTACGCCGAAGGG;R：AGTCCGATCGCCTATGAGGA	897	56
PeVYV	F：GACGACGAAATGGAGGC;R：CATTAATCTGCGAAGCCCG	361	51
CFEV 1	F：TGTGCGTGAAGGGTGTTTCT;R：GCGGTCATGCGAGTAGTGAA	568	56
PCV 2	F：CACGTGTGAGGGCATTTGAC;R：GTCCGACACCGTATGCCTTT	717	57
TMGMV	F：TTCCGCTTATGCAGATCCTGT;R：GACGAACAACCACTGCTGAT	523	54
PepMoV	F：GGACTTCTGTTTGTCGGTGAT;R：AGATGTCTGAAGTGAGTCATCAG	385	50
PVY	F：AGCAAAGCAGCATCCAGTCA;R：GTACTGATGCCACCGTCCAA	670	56
TMV	F：GTTCTTGTCATCTGCGTGGG;R：GCTCTCGAAAGAGCTCCGAT	406	57

采样地 Sampling site	sRNA 原始读数 Number of sRNA raw reads	高质量sRNA读数 Number of sRNA clean reads	重叠群Contigs		高质量siRNA序列 siRNA clean reads
采样地 Sampling site	sRNA 原始读数 Number of sRNA raw reads	高质量sRNA读数 Number of sRNA clean reads	重叠群N50/bp Contigs_N50	重叠群数 Number of contigs	数量 Number	占比/% Percentage
横县Hengxian	20 895 583	20 380 089	135	738	8 630 968	42.35
钦州Qinzhou	20 275 068	19 860 840	108	576	7 688 131	38.71
五塘Wutang	19 934 894	19 044 126	92	953	6 328 363	33.23
柳江Liujiang	19 576 663	19 207 270	132	685	6 088 705	31.70
桂林Guilin	19 742 187	19 083 857	86	505	2 383 574	12.49
北海Beihai	20 170 123	18 339 543	196	400	9 448 533	51.52
武鸣Wuming	17 175 774	16 804 826	141	433	10 212 293	60.77
百色Baise	20 390 152	19 925 555	84	609	7 884 542	39.57
凭祥Pingxiang	19 234 300	18 607 934	116	474	3 232 198	17.37

采样地 Sampling site	sRNA 原始读数 Number of sRNA raw reads	高质量sRNA读数 Number of sRNA clean reads	重叠群Contigs		高质量siRNA序列 siRNA clean reads
采样地 Sampling site	sRNA 原始读数 Number of sRNA raw reads	高质量sRNA读数 Number of sRNA clean reads	重叠群N50/bp Contigs_N50	重叠群数 Number of contigs	数量 Number	占比/% Percentage
横县Hengxian	20 895 583	20 380 089	135	738	8 630 968	42.35
钦州Qinzhou	20 275 068	19 860 840	108	576	7 688 131	38.71
五塘Wutang	19 934 894	19 044 126	92	953	6 328 363	33.23
柳江Liujiang	19 576 663	19 207 270	132	685	6 088 705	31.70
桂林Guilin	19 742 187	19 083 857	86	505	2 383 574	12.49
北海Beihai	20 170 123	18 339 543	196	400	9 448 533	51.52
武鸣Wuming	17 175 774	16 804 826	141	433	10 212 293	60.77
百色Baise	20 390 152	19 925 555	84	609	7 884 542	39.57
凭祥Pingxiang	19 234 300	18 607 934	116	474	3 232 198	17.37

病毒 Virus	桂南Southern Guangxi						桂中 Central Guangxi	桂西 Western Guangxi	桂北 Northern Guangxi	总检出率 Total detection rate
病毒 Virus	横县 Hengxian	钦州 Qinzhou	五塘 Wutang	北海 Beihai	武鸣 Wuming	凭祥 Pingxiang	柳江 Liujiang	百色 baise	桂林 Guilin	总检出率 Total detection rate
ChiVMV	28.57	25.00	100.00	100.00	75.00	83.33	100.00	50.00	33.33	66.67
PVMV	100.00	100.00	90.00	100.00	75.00	33.33	-	-	16.67	62.67
CMV	-	50.00	20.00	100.00	75.00	100.00	16.67	100.00	16.67	41.33
CFEV 1	14.29	25.00	20.00	28.57	-	33.33	100.00	50.00	-	32.00
PCV 2	71.43	-	20.00	-	50.00	-	-	25.00	50.00	26.67
PMMoV	28.57	25.00	40.00	-	-	-	33.33	50.00	50.00	22.67
PeVYV	42.86	-	-	28.57	25.00	33.33	-	100.00	-	21.33
CHiRSV	28.57	-	-	-	25.00	-	66.67	50.00	-	21.33
PepMoV	28.57	-	80.00	-	-	-	-	-	-	16.00
TMGMV	42.86	-	-	-	-	-	-	-	-	8.00
PVY	-	-	-	-	-	-	-	-	-	0.00
TMV	-	-	-	-	-	-	-	-	-	0.00