基于种特异性COI标记的西花蓟马温室品系和羽扇豆品系一步双重快速鉴定技术

doi:10.3864/j.issn.0578-1752.2019.16.007

中国农业科学 ›› 2019, Vol. 52 ›› Issue (16): 2809-2823.doi: 10.3864/j.issn.0578-1752.2019.16.007

基于种特异性COI标记的西花蓟马温室品系和羽扇豆品系一步双重快速鉴定技术

张蓉¹,王玉生¹,杨丽梅³,万方浩^1,²,张桂芬^1,²()

1 中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室/农业农村部作物有害生物综合治理重点实验室,北京 100193
2 农业农村部外来入侵生物预防与控制研究中心,北京 100193
3 北京市大兴区榆垡镇农业技术推广站,北京 102602

收稿日期:2019-03-27 接受日期:2019-06-06 出版日期:2019-08-16 发布日期:2019-08-21
通讯作者: 张桂芬
作者简介:张蓉,E-mail：zhangrong0120@163.com。
基金资助:
国家自然科学基金(31572067);国家重点研发计划(2017YFC1200600)

One-Step Duplex Rapid Identification Technique of Frankliniella occidentalis Greenhouse and Lupin Races Based on Species-Specific COI Marker

ZHANG Rong¹,WANG YuSheng¹,YANG LiMei³,WAN FangHao^1,²,ZHANG GuiFen^1,²()

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests/Key Laboratory of Integrated Pest Management of Crop, Ministry of Agriculture and Rural Affairs, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
2 Center for Management of Invasive Alien Species, Ministry of Agriculture and Rural Affairs, Beijing 100193
3 Agricultural Technology Extension Station of Yufa, Daxing, Beijing 102602

Received:2019-03-27 Accepted:2019-06-06 Online:2019-08-16 Published:2019-08-21
Contact: GuiFen ZHANG

摘要/Abstract

摘要：

【背景】西花蓟马（Frankliniella occidentalis）是世界性农业和园艺作物害虫,也是我国重要的对外对内检疫对象。西花蓟马具有两个品系,即温室品系（greenhouse race,GR）和羽扇豆品系（lupin race,LR）,二者在寄主范围、抗药性、生存环境等方面均具有明显差异,但外部形态相似,无法准确鉴别。【目的】以西花蓟马温室品系和羽扇豆品系为靶标,以田间常见的其他14种蓟马为参照,采用基于线粒体DNA细胞色素C氧化酶亚基I（mitochondrial DNA cytochrome c oxidase subunit I,mtDNA COI）基因的种特异性（species-specific COI,SS-COI）PCR法,研究一步双重品系快速检测技术。【方法】采用mtDNA COI基因通用引物获得西花蓟马温室品系和羽扇豆品系以及其他常见蓟马的COI序列,根据测序结果以及数据库已公开数据,设计筛选获得可同时扩增两个品系的特异性组合引物（TF6/GR32/LR12）,其中TF6为品系通用上游引物,GR32为温室品系特异性下游引物,LR12为羽扇豆品系特异性下游引物,其扩增片段大小分别为温室品系362 bp、羽扇豆品系541 bp;对组合引物比例和退火温度进行优化;同时,对组合引物的种/品系特异性和灵敏性/检测阈值进行检验。【结果】当组合引物TF6/GR32/LR12比例为1.0/0.2/0.8、退火温度为44℃时,扩增效果最好。种/品系特异性检测结果证实,该检测技术仅对西花蓟马温室品系和羽扇豆品系具有扩增能力,对田间常见的其他14种蓟马包括花蓟马（Frankliniella intonsa）、禾花蓟马（Frankliniella tenuicornis）、黄胸蓟马（Thrips hawaiiensis）、大蓟马（Thrips major）、棕榈蓟马（Thrips palmi）、烟蓟马（Thrips tabaci）、普通大蓟马（Megalurothrips usitatus）、豆喙蓟马（Mycterothrips glycines）、草木樨近绢蓟马（Sussericothrips melilotus）、蔗腹齿蓟马（Fulmekiola serrata）、稻简管蓟马（Haplothrips aculeatus）、榕端宽管蓟马（Mesothrips jordani）、榕母管蓟马（Gynaikothrips ficorum）和横纹蓟马（Aeolothrips fasciatus）等不具有扩增能力。灵敏性检测结果显示,该组合引物不仅对单一品系或混合品系的成虫具有良好的扩增效果,对单粒卵、1龄和2龄幼虫以及预蛹和蛹均具有同样的扩增效能;对温室品系的最低检测阈值为35.90 pg·μL ^-1,相当于1/10 240头雌成虫,对羽扇豆品系的最低检测阈值为146.95 pg·μL ^-1,相当于1/2 560头雌成虫;同时,对来自不同地域的同一品系不同单倍型的成虫亦具有良好的扩增效能。【结论】建立的技术体系完全可以用于西花蓟马品系的快速鉴定及检疫监管,对有效阻截其进一步传播扩散及靶向防控措施制定意义重大。

关键词: 西花蓟马温室品系, 西花蓟马羽扇豆品系, 一步双重PCR, SS-COI标记, 品系特异性组合引物, 快速鉴定

Abstract:

【Background】Frankliniella occidentalis is one of the most important pests infesting agricultural and horticultural crops worldwide, also it is a national- and international-wide important quarantine pest. F. occidentalis has two races, i.e., greenhouse race (GR) and lupin race (LR). There are significant differences between the two races in host plant species, pesticide resistance, living environments, etc. However, it is difficult to identify these two races because of their high similarity in morphological characteristics.【Objective】In this study, a one-step duplex PCR procedure for rapid identification of F. occidentalis greenhouse race and lupin race was developed by using species-specific COI (SS-COI) marker based on mitochondrial DNA cytochrome c oxidase subunit I (mtDNA COI) gene, and other 14 common thrips species in the field were used as the control.【Method】The COI sequences of F. occidentalis greenhouse and lupin races, and other 14 thrips species were amplified using mtDNA COI gene universal primers. The race-specific primer combination system, TF6/GR32/LR12 (TF6, the universal forward primer; GR32, the greenhouse race-specific reverse primer; LR12, the lupin race-specific reverse primer), for one-step duplex amplification of the two races was established. The fragments amplified by the combined primers were 362 bp for the greenhouse race and 541 bp for the lupin race, respectively. The system and conditions (e.g., annealing temperature) were optimized. The species/race specificity and sensitivity/ detection threshold of the combined primers were evaluated.【Result】When the ratio of the combined primers (TF6/GR32/LR12) was 1.0/0.2/0.8 and the annealing temperature was 44℃, the amplification effect was the best. Species/race-specific tests performed with this set of combined primers indicated that all F. occidentalis greenhouse race and lupin race specimens were detected positively and no cross reaction with other 14 thrips species, including Frankliniella intonsa, Frankliniella tenuicornis, Thrips hawaiiensis, Thrips major, Thrips palmi, Thrips tabaci, Megalurothrips usitatus, Mycterothrips glycines, Sussericothrips melilotus, Fulmekiola serrate, Haplothrips aculeatus, Mesothrips jordani, Gynaikothrips ficorum, and Aeolothrips fasciatus, was identified. The system was tested on individual male and female adults, individual prepupae and pupae, individual 1st and 2nd instar larvae, and even single egg, and demonstrated to be applicable for all these stages of greenhouse and lupin races of F. occidentalis, either in single race or two races mixed together. The detection threshold for greenhouse race was 35.90 pg·μL ^-1, which was equivalent to 1/10 240 of a whole female adult. The detection threshold for lupin race was 146.95 pg·μL ^-1, which was equivalent to 1/2 560 of a whole female adult. Meanwhile, the system worked well in testing individuals from different geographic populations with different haplotypes within greenhouse race or lupin race of the thrips. 【Conclusion】The one-step duplex PCR system developed here for rapid identification of F. occidentalis greenhouse and lupin races could be useful in port quarantine and interception in national and international transportation of agricultural products, and should be significant in blocking further spreading and developing targeted prevention and control measures of F. occidentalis.

Key words: Frankliniella occidentalis greenhouse race, F. occidentalis lupin race, one-step duplex PCR, SS-COI marker, race-specific combined primer set, rapid identification

张蓉,王玉生,杨丽梅,万方浩,张桂芬. 基于种特异性COI标记的西花蓟马温室品系和羽扇豆品系一步双重快速鉴定技术[J]. 中国农业科学, 2019, 52(16): 2809-2823.

ZHANG Rong,WANG YuSheng,YANG LiMei,WAN FangHao,ZHANG GuiFen. One-Step Duplex Rapid Identification Technique of Frankliniella occidentalis Greenhouse and Lupin Races Based on Species-Specific COI Marker[J]. Scientia Agricultura Sinica, 2019, 52(16): 2809-2823.

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图/表 11

表1

蓟马样本信息"

编号 No.	物种名称 Species	地点 Location	寄主 Host plant	时间 Date	采集人 Collector
1	西花蓟马温室品系 F. occidentalis greenhouse race	北京延庆,室内饲养 Yanqing, Beijing; indoor rearing	市售芸豆 Phaseolus vulgaris; marketed	2017-07	魏书军 WEI ShuJun
2	西花蓟马羽扇豆品系 F. occidentalis lupin race	北京延庆,室内饲养 Yanqing, Beijing; indoor rearing	市售芸豆 P. vulgaris; marketed	2017-07	魏书军 WEI ShuJun
3	花蓟马 F. intonsa	宁夏吴忠 Wuzhong, Ningxia	月季 Rosa chinensis	2016-08	田虎 TIAN Hu
4	禾花蓟马 F. tenuicornis	甘肃张掖 Zhangye, Gansu	葱 Allium fistulosum	2016-08	田虎 TIAN Hu
5	黄胸蓟马 T. hawaiiensis	山东烟台 Yantai, Shandong	未知种 Unidentified species	2016-11	田虎 TIAN Hu
6	大蓟马 T. major	青海西宁 Xining, Qinghai	月季 R. chinensis	2016-08	田虎 TIAN Hu
7	棕榈蓟马 T. palmi	浙江温州 Wenzhou, Zhejiang	西瓜 Citrullus lanatus	2018-07	薛延韬 XUE YanTao
8	烟蓟马 T. tabaci	贵州安顺 Anshun, Guizhou	茼蒿 Chrysanthemum coronarium	2018-04	杜素洁 DU SuJie
9	普通大蓟马 M. usitatus	广东河源 Heyuan, Guangdong	芸豆 P. vulgaris	2017-12	毛苗苗 MAO MiaoMiao
10	豆喙蓟马 M. glycines	山东滨州 Binzhou, Shandong	大豆 Glycine max	2018-06	薛延韬 XUE YanTao
11	草木樨近绢蓟马 S. melilotus	北京西城 Xicheng, Beijing	白三叶 Trifolium repens	2018-07	张桂芬 ZHANG GuiFen
12	蔗腹齿蓟马 F. serrata	山东青岛 Qingdao, Shandong	白茅 Imperata cylindrica	2018-07	薛延韬 XUE YanTao
13	稻简管蓟马 H. aculeatus	山东青岛 Qingdao, Shandong	芦苇 Phragmites australis	2018-07	薛延韬 XUE YanTao
14	榕端宽管蓟马 M. jordani	广东深圳 Shenzhen, Guangdong	榕树 Ficus microcarpa	2018-07	薛延韬 XUE YanTao
15	榕母管蓟马 G. ficorum	福建漳州 Zhangzhou, Fujian	榕树 F. microcarpa	2018-07	薛延韬 XUE YanTao
16	横纹蓟马 A. fasciatus	天津滨海新区 Binhai New District, Tianjin	大豆 G. max	2018-06	薛延韬 XUE YanTao

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品系 Race	引物名称 Primer name	碱基序列 Primer sequence (5′-3′)	扩增片段长度 Fragment length (bp)
温室品系 Greenhouse race	TF6/GR32	CGACTTAATAACATAAGATTTT/ GATGTATCTAAGTCTCGGTCT	362
羽扇豆品系 Lupin race	TF6/LR12	CGACTTAATAACATAAGATTTT/ GCATAGCATAGATTAGTCCC	541

采样地点 Location	种群代码 Population code	寄主植物 Host plant	采集时间 Collection date	测序个体数量 Number of sequenced
宁夏银川 Yinchuan, Ningxia	YC	美人蕉 Canna indica	2015-08	21
陕西宝鸡 Baoji, Shaanxi	BJI	月季 R. chinensis	2015-08	18
山东烟台 Yantai, Shandong	YT	蜀葵 Althaea rosea	2015-07	25
甘肃敦煌 Dunhuang, Gansu	DH	月季 R. chinensis	2015-08	8
吉林四平 Siping, Jilin	SP	美人蕉 C. indica	2014-09	2
吉林长春 Changchun, Jilin	CC	金盏菊 Calendula officinalis	2014-09	9
云南临沧 Lincang, Yunnan	LC	白三叶 T. repens	2015-04	12
甘肃兰州 Lanzhou, Gansu	LZ	辣椒 Capsicum annuum	2015-08	21
北京延庆 Yanqing, Beijing	YQ	辣椒 C. annuum	2014-07	8
河北廊坊 Langfang, Hebei	LF	辣椒 C. annuum	2015-07	3
合计Total	10			127

种群 Population	单倍型 Haplotype
	温室品系Greenhouse race			羽扇豆品系Lupin race
	GHap-1	GHap-2	GHap-3		LHap-1	LHap-2
YC	14	1			5	1
BJI	15	1			2
YT	20	3	1		1
DH	7	1
SP	1				1
CC	6				3
LC	12
LZ	19	2
YQ	7	1
LF	3
总计 Total	104	9	1		12	1

基于种特异性COI标记的西花蓟马温室品系和羽扇豆品系一步双重快速鉴定技术

One-Step Duplex Rapid Identification Technique of Frankliniella occidentalis Greenhouse and Lupin Races Based on Species-Specific COI Marker

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